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Abstract:

The present invention provides use of the compounds represented by
formula (1): wherein, R1, R2, R3 and R4 are as
defined in the specification, in all their stereo isomeric and tautomeric
forms and mixtures thereof in all ratios, and their pharmaceutically
acceptable salts, pharmaceutically acceptable solvates, pharmaceutically
acceptable polymorphs and prodrugs and pharmaceutical compositions
containing them for treatment of inflammatory disorders mediated by one
or more cytokines selected from Tumor Necrosis Factor-alpha
(TNF-α), interferon-γ (IFN-γ) and interleukins such as
IL-1β, IL-2, IL-6, and IL-8. The present invention also relates to a
pharmaceutical composition adapted for use in the treatment of
inflammatory disorders. The present invention further provides a method
of treatment of inflammatory disorders by administering a therapeutically
effective amount of the said compound of formula (1) or its
pharmaceutical composition, to a mammal in need thereof.
##STR00001##

Claims:

1-26. (canceled)

27. A method for the treatment of an inflammatory disorder, comprising
administering to a subject in need thereof a therapeutically effective
amount of a compound of the following formula (1), or a stereoisomeric
form, or a tautomeric form, or a pharmaceutically acceptable salt
thereof, ##STR00061## wherein, R1 is selected from halogen,
hydroxy, alkoxy, --O(CO)R13, --SR14, and --NR14R15;
R2 is hydrogen; or optionally R1 is absent and R2 is
═O; R3 is alkyl; R4 is selected from the following
formulae: ##STR00062## R5 is selected from hydroxy or alkoxy;
R6 is selected from hydrogen, hydroxy, alkyl, and alkoxy; R7 is
selected from hydrogen, alkyl, and --(CO)R16; R8 is selected
from hydroxy or alkoxy; R9 is selected from hydroxy, alkyl, alkoxy,
aryl, aralkyl, aryloxy, benzyloxy, heterocyclyl, --O-heterocyclyl,
--OCH2COOR17, and --OCH2COR18; R10 is selected
from halogen, hydroxy, alkoxy, --SR14, --NR14R15, and
--O(CO)R19; R11 is selected from hydrogen or halogen; R12
is selected from hydrogen, halogen, and hydroxy; R13 is selected
from alkyl or aryl; R14 is selected from hydrogen, alkyl, aralkyl,
aryl, and heterocyclyl; R15 is selected from hydrogen or alkyl;
R16 is selected from alkyl or aryl; R17 is selected from
hydrogen, or alkyl; R18 is selected from alkyl,
--NHCH2R20, aryl, and heterocyclyl; R19 is selected from
alkyl, aralkyl, aryl, and heterocyclyl; and R20 is selected from
hydrogen, alkyl, aryl, and heterocyclyl; where alkyl is unsubstituted or
substituted by one or two of the same or different groups selected from:
hydroxy, halogen, amino, hydroxyalkyl, alkoxy, aryl, aryloxy, and
heterocyclyl; alkoxy is unsubstituted or substituted by one or two of the
same or different groups selected from: halogen, hydroxy, alkyl, and
hydroxyalkyl; aryl is unsubstituted or substituted by one or two of the
same or different groups selected from: halogen, hydroxy, amino, alkyl,
hydroxyalkyl, alkoxy, aryl, and heterocyclyl; heterocyclyl is
unsubstituted or substituted by one or two of the same or different
groups selected from: halogen, hydroxy, amino, alkyl, hydroxyalkyl,
alkoxy, aryl, and heterocyclyl.

28. The method according to claim 27, wherein in the compound of formula
(1) R1 is selected from halogen, hydroxy, alkoxy, --O(CO)R13,
--SR14, and --NR14R15; R2 is hydrogen; R3 is
alkyl; R4 is selected from the following formulae: ##STR00063##
R5 is selected from hydroxy, or alkoxy; R6 is selected from
hydrogen, hydroxy, alkyl, and alkoxy; R7 is selected from hydrogen,
alkyl, and --(CO)R16; R8 is selected from hydroxy, or alkoxy;
R9 is selected from hydroxy, alkyl, alkoxy, aryl, aralkyl, aryloxy,
benzyloxy, heterocyclyl, --O-heterocyclyl, --OCH2COOR17, and
--OCH2COR18; R10 is selected from halogen, hydroxy,
alkoxy, --SR14, --NR14R15, and --O(CO)R19; R11
is selected from hydrogen, or halogen; R12 is selected from
hydrogen, halogen, and hydroxy; R13 is selected from alkyl, or aryl;
R14 is selected from hydrogen, alkyl, aralkyl, aryl, and
heterocyclyl; R15 is selected from hydrogen, or alkyl; R16 is
selected from alkyl, or aryl; R17 is selected from hydrogen, or
alkyl; R18 is selected from alkyl, --NHCH2R20, aryl, and
heterocyclyl; R19 is selected from alkyl, aralkyl, aryl, and
heterocyclyl; and R20 is selected from hydrogen, alkyl, aryl, and
heterocyclyl; where alkyl is unsubstituted or substituted by one or two
of the same or different groups selected from: hydroxy, halogen, amino,
hydroxyalkyl, alkoxy, aryl, aryloxy, and heterocyclyl; alkoxy is
unsubstituted or substituted by one or two of the same or different
groups selected from: halogen, hydroxy, alkyl, and hydroxyalkyl; aryl is
unsubstituted or substituted by one or two of the same or different
groups selected from: halogen, hydroxy, amino, alkyl, hydroxyalkyl,
alkoxy, aryl, and heterocyclyl; heterocyclyl is unsubstituted or
substituted by one or two of the same or different groups selected from:
halogen, hydroxy, amino, alkyl, hydroxyalkyl, alkoxy, aryl, and
heterocyclyl.

29. The method according to claim 27, wherein the compound of formula (1)
is selected from: ##STR00064## ##STR00065## ##STR00066## or a
stereoisomeric form, tautomeric form, pharmaceutically acceptable salt or
thereof

31. The method according to claim 27, wherein the inflammatory disorder
is mediated by one or more inflammatory cytokines selected from Tumor
Necrosis Factor-alpha (TNF-.alpha.), interferon-.gamma. (IFN-.gamma.) and
interleukins (IL-1.beta., IL-2, IL-6 and IL-8).

33. The method according to claim 31, wherein the inflammatory disorder
mediated by interleukins (IL-1.beta., IL-2, IL-6 and IL-8) is selected
from the group consisting of rheumatoid arthritis, osteoarthritis and
autoimmune conditions.

34. The method according to claim 31, wherein the inflammatory disorder
mediated by interferon-.gamma. (IFN-.gamma.) is selected from the group
consisting of rheumatoid arthritis, osteoarthritis and autoimmune
conditions.

36. The method according to claim 35, wherein the inflammatory disorder
is rheumatoid arthritis.

37. The method according to claim 35, wherein the inflammatory disorder
is ulcerative colitis.

38. A method for monitoring drug response in a patient with an
inflammatory disorder treated with a compound of formula (1) comprising
determining the expression of one or more of genes selected from
CEBPα, CEBPβ, CEBPδ, IL-1.beta., IL-6, GBP-1, MMP 13,
MyD88, BCL2 and cMyc in a test sample obtained from the patient treated
with said compound of formula (1) and comparing it to the expression of
the same one or more of CEBPα, CEBPβ, CEBPδ, IL-1.beta.,
IL-6, GBP-1, MMP 13, MyD88, BCL2 and cMyc in a sample obtained from the
patient before treatment with the compound of formula (1).

39. The method according to claim 38, wherein a change in the expression
of the one or more of genes selected from CEBPα, CEBPβ,
CEBPδ, IL-1.beta., IL-6, GBP-1, MMP 13, MyD88, BCL2 and cMyc after
treatment with the compound of formula (1) is indicative of a drug
response.

40. The method according to claim 38, wherein the expression of one or
more of genes selected from CEBPα, CEBPβ, CEBPδ,
IL-1.beta., IL-6, GBP-1, MMP 13, MyD88, BCL2 and cMyc is down-regulated
after treatment with the compound of formula (1).

41. The method according to claim 38, wherein the compound of formula (1)
is ##STR00069## or a stereoisomeric form, or a tautomeric form, or a
pharmaceutically acceptable salt, thereof.

42. The method according to claim 38, wherein the inflammatory disorder
is mediated by CREB pathway.

Description:

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is related to our copending PCT application
entitled: "MACROCYCLIC LACTONE DERIVATIVES FOR THE TREATMENT OF CANCER",
filed on the same date as the present application.

FIELD OF THE INVENTION

[0002] The present invention relates to the use of macrocyclic lactone
derivatives, and pharmaceutical compositions containing them for the
treatment of inflammatory disorders mediated by one or more cytokines
selected from Tumor Necrosis Factor-alpha (TNF-α),
interferon-γ (IFN-γ) and interleukins such as IL-1β,
IL-2, IL-6, and IL-8.

BACKGROUND OF THE INVENTION

[0003] Inflammation is the body's biological response to infection or
tissue damage. Under physiological conditions, it is the primary means by
which the body fights off invading pathogens and heals the injured
tissue. An aberrant inflammatory response can lead to tissue damage and
destruction. Chronic uncontrolled inflammation can lead to diseases such
as rheumatoid arthritis, osteoarthritis, psoriasis, atherosclerosis,
asthma and inflammatory bowel disease (including ulcerative colitis and
Crohn's disease).

[0004] Rheumatoid arthritis (RA), an autoimmune disorder, is a chronic,
systemic, articular inflammatory disease. In addition to joint swelling
and pain caused by the inflammatory process, the ultimate hallmark of RA
is joint destruction. Indeed, in RA, the normally thin synovial lining of
joints is replaced by an inflammatory and highly vascularized, invasive
fibrocollagenase tissue (pannus), which is destructive to both cartilage
and bone. RA produces its prominent manifestations in the synovial
joints. Cartilage destruction in RA is linked to aberrant production of
pro-inflammatory cytokines [including tumor necrosis factor-α
(TNF-α), interleukin-6 (IL-6) and other interleukins (IL-1β
and IL-8)] and growth factor expression in the affected joints.

[0005] Psoriasis is an auto-immune/inflammatory disease and although the
etiology of psoriasis remains unknown, it is well established that
T-cells play a destructive role in psoriasis. Upon getting activated by
antigen-presenting cells in the lymph node draining to the skin, T-cells
migrate into the skin. In the psoriatic lesions, T-cells release type 1
cytokines [e.g., interleukin-2 (IL-2) and interferon-γ
(IFN-α)] and stimulate the neighboring leukocytes. The secreted
pro-inflammatory mediators (e.g., TNF-α) drive the
hyperproliferation of keratinocytes and, thereby, augment the
inflammatory damage in the psoriatic plaque.

[0006] Inflammatory bowel disease (IBD) is a group of disorders that cause
inflammation of the intestines. The inflammation lasts for a long time
and usually relapses. The two major types of IBD are Crohn's disease and
ulcerative colitis. Crohn's disease occur when the lining and wall of the
intestines becomes inflamed resulting in the development of ulcers.
Although Crohn's disease can occur in any part of the digestive system,
it often occurs in the lower part of the small intestine where it joins
the colon. Ulcerative colitis is a chronic auto-immune/inflammatory
disease of unknown etiology afflicting the large intestine. Neither the
initiating event nor the sequence of propagating events that lead to and
sustain colitis have been fully elucidated. Nevertheless, it is
well-established that a dysfunctional immune-response involving
components of normal gastrointestinal gram-negative bacteria and
increased expression of pro-inflammatory cytokines, chemokines,
endothelial cell adhesion molecules (ECAMs) and enhanced leukocyte
infiltration into colonic interstitium, play a key role in the
pathogenesis of colitis. The course of the disease may be continuous or
relapsing, mild or severe. Signs and symptoms of the disease include
cramping, lower abdominal pain, rectal bleeding, and frequent, loose
discharges consisting mainly of blood, pus, and mucus with scanty fecal
particles.

[0007] The first line of treatment for inflammatory disorders involves the
use of non-steroidal anti-inflammatory drugs (NSAIDs) e.g. ibuprofen,
naproxen to alleviate symptoms such as pain. However, despite the
widespread use of NSAIDs, many individuals cannot tolerate the doses
necessary to treat the disorder over a prolonged period of time as NSAIDs
are known to cause gastric erosions. Moreover, NSAIDs merely treat the
symptoms of disorder and not the cause. When patients fail to respond to
NSAIDs, other drugs such as methotrexate, gold salts, D-penicillamine and
corticosteroids are used. These drugs also have significant toxic
effects. An increased understanding of the molecular events leading to
inflammatory disorders has led to novel approaches for targeting the
pathogenesis. Although, the entire sequence of pathobiological events
that leads to and sustains various inflammatory disorders is yet to be
completely elucidated, it is well established that a milieu of
pro-inflammatory cytokines (e.g., TNF-α, IL-6 and IL-1β) play
an important role in the pathogenesis of these inflammatory disorders.
Indeed, several studies utilizing animal models of inflammation (e.g.,
RA/colitis/psoriasis) and, more importantly clinically relevant studies
of patients with active RA/colitis/psoriasis, have demonstrated that
these cytokines are expressed in high-levels both in the inflamed tissue
as well as the peripheral blood of patients. These pro-inflammatory
mediators induce/up-regulate the expression of adhesion molecules on the
microvascular endothelium that leads to recruitment and infiltration of
inflammatory leukocytes. Furthermore, in RA settings these cytokines, by
virtue of activating osteoclasts, participate in perpetuating
inflammation and eventually lead to cartilage degradation and bone
erosion. TNF-α, a pleiotropic cytokine, is produced mainly by
macrophages. TNF-α demonstrates beneficial as well as pathological
activities. It has both growth stimulating effects and growth inhibitory
properties, besides being self-regulatory. TNF-α induces the
expression of a variety of genes that contribute to various
auto-immune/inflammatory disorders.

[0008] Although TNF-α plays a critical role in innate and acquired
immune responses, an increase in the production of TNF-α can
produce pathological changes resulting in chronic inflammation and tissue
damage. TNF-α has been shown to play a crucial role in the
pathogenesis of many chronic inflammatory disease such as rheumatoid
arthritis, juvenile rheumatoid arthritis, psoriatic arthritis,
osteoarthritis, refractory rheumatoid arthritis, chronic non-rheumatoid
arthritis, osteoporosis/bone resorption, coronary heart disease,
vasculitis, inflammatory bowel disease, ulcerative colitis, Crohn's
disease, adult respiratory distress syndrome, diabetes, psoriasis, skin
delayed type hypersensitivity disorders and Alzheimer's disease. As a
specific example, it is well recognized that TNF-α is at the apex
of the pro-inflammatory cytokine network in RA. Indeed, it controls the
production of other cytokines and orchestrates the
inflammatory/immune-response in the synovium. Consistent with this,
transgenic mice bearing a de-regulated TNF gene spontaneously develop
chronic inflammatory arthritis, and neutralization of TNF-α
decreases the incidence and severity of inflammatory arthritis in animal
models of RA. These, and several other studies, have demonstrated that
TNF-α is an attractive therapeutic target in controlling the
aberrant immune/inflammatory response in RA (and also other diseases such
as psoriasis and IBD). Most importantly, clinically approved therapies
for treating active RA, psoriasis and IBD include TNF-α inhibitors
[etanercept (Enbrel), infliximab (Remicade) and adalimumab (Humira)]. In
spite of the widespread use of these TNF-α inhibitors, up to 50% of
patients treated with TNF blockers fail to improve disease status
significantly. Furthermore, while the goal of therapy (in RA patients) is
to achieve disease remission and stop joint destruction, existing
biologics targeting TNF-α have been shown to stop disease
progression in a proportion of, and not all, RA patients.

[0011] The therapeutic success of biological inhibitors for TNF-α
prompted the development of biological modulators for other targets.
Recently, tociluzimab a humanized antibody that binds to both soluble and
membrane bound IL-6 receptor has shown exceptional therapeutic efficacy
in clinical trials for rheumatoid arthritis. Tocilizumab is approved for
treating patients with active RA in Japan and has also gained approval of
the FDA's advisory board. Based on this data, interleukin-6 is
recommended as a new therapeutic target (Arthritis Research and Therapy,
8(suppl 2), S5, (2006)). Separately, the use of these biologic agents is
associated with severe limitations (e.g., parenteral route of
administration, high cost of therapy, risk of opportunistic infections,
induction of allergic reactions, activation of latent tuberculosis,
increased risk of cancer, risk for worsening congestive heart disease).
As such, there is an unmet need for small molecule inhibitors of
IL-6/TNF-α that would have the same effect as biological agents but
without the undesirable side effects.

[0012] Intervention of biological activity of IL-6 can be achieved by
blocking IL-6 production and/or neutralizating IL-6 (Annals of the
Rheumatic Diseases, 59 (suppl 1), i21-i27 (2000)). The etiology of
rheumatoid arthritis, inflammatory disorders and other inflammatory
conditions is also characterized by uninhibited T-cell proliferation
(Arthritis & Rheumatism, 35: 729-735, (1992)). One class of compounds
that has received increased attention is compounds inhibiting T-cell
proliferation. Limiting T cell activation restricts T cell proliferation
as well as the production of the Th1 cytokines, which are implicated in
activation of the monocyte macrophage system in the rheumatoid or
synovial milieu (Arthritis Research, 4 (suppl 3):S197-S211, (2002)).

[0013] T cell activation is marked by the expression of specific proteins
that aid in their effect or functions. Among the first proteins to be
expressed are interleukin-2 (IL-2) and IL-2 receptor alpha subunit. IL-2
is a potent T cell mitogen, which is required for T cell proliferation.
IL-2 signaling is required for T cells to initiate the immune response.
IL-2 is a potent T cell growth cytokine, which, in T cell activation,
acts in an autocrine fashion to promote the growth, proliferation and
differentiation of the T cell which has been recently stimulated by
antigen. Indeed, T cells that receive inappropriate signaling become
anergic i.e. they become inactive. This is accomplished by making the T
cell unable to synthesize IL-2. This renders them potentially inert to
any antigenic stimulation they might receive in the future. Clinically
compounds such as cyclosporin, FK506 and rapamycin are known to induce
anergy to continuous antigenic stimulus by blocking this IL-2 signaling
via distinct pathways.

[0016] The compounds described in the present invention inhibit T-cell
proliferation and block production of the cytokines. These effects may be
contributing towards their therapeutic efficacy.

[0017] Transcriptional coactivators have crucial roles in eukaryotic
transcription. One of the factors that can activate transcription factors
in macrophages is bacterial lipopolysaccharide (LPS). Bacterial endotoxin
such as LPS is known to be one of the inducers of macrophagic activation.
Activation of macrophages is involved in augmentation of several
inflammatory conditions; e.g., rheumatoid arthritis, inflammatory bowel
disease, sepsis and other diseases. LPS activation of macrophages
triggers Toll-like receptor 4 (TLR4). TLR4 is a protein that in humans is
encoded by the TLR4 gene. TLR4 signalling and activation of TLRs is
associated with induction of pro-inflammatory gene expression.

[0020] The present invention relates to the use of macrocyclic lactone
derivatives for the treatment of an inflammatory disorder mediated by one
or more cytokines selected from Tumor Necrosis Factor-alpha
(TNF-α), interferon-γ (IFN-γ) and interleukins such as
IL-1β, IL-2, IL-6, and IL-8.

[0021] According to a further aspect, there is provided the use of
compound of formula (1) (as provided herein below), for the treatment of
an inflammatory disorder mediated by one or more cytokines selected from
Tumor Necrosis Factor-alpha (TNF-α), interferon-γ
(IFN-γ) and interleukins such as IL-1, IL-2, IL-6, and IL-8.

[0022] According to another aspect of the present invention, there are
provided pharmaceutical compositions including one or more compounds of
formula (1) as active ingredient, for the treatment of an inflammatory
disorder mediated by one or more cytokines selected from Tumor Necrosis
Factor-alpha (TNF-α), interferon-γ (IFN-γ) and
interleukins such as IL-1β, IL-2, IL-6, and IL-8.

[0023] According to another aspect of the present invention, there is
provided a method for the treatment of an inflammatory disorder mediated
by one or more cytokines selected from Tumor Necrosis Factor-alpha
(TNF-α), interferon-γ (IFN-γ) and interleukins such as
IL-1β, IL-2, IL-6, and IL-8 the method including administering to a
mammal in need thereof, a therapeutically effective amount of one or more
compounds of general formula (1).

[0024] According to another aspect of the present invention, there are
provided methods for the manufacture of medicaments including one or more
compounds of formula (1) which are useful for the treatment of an
inflammatory disorder mediated by one or more cytokines selected from
Tumor Necrosis Factor-alpha (TNF-α), interferon-γ
(IFN-γ) and interleukins such as IL-1β, IL-2, IL-6, and IL-8.

[0025] According to still another aspect of the invention, there are
provided methods for the treatment of an inflammatory disorder by
down-regulating one or more genes selected from BCL2, CEBPα,
CEBPβ, CEBPδ, IL-1β, IL-6, cMyc, GBP-1, MMP13 and MyD88.

[0026] According to another aspect of the invention, there is provided a
method for monitoring drug response in a patient with an inflammatory
disorder treated with a compound of formula (1), comprising determining
the expression of one or more genes selected from CEBPα,
CEBPβ, CEBPδ, IL-1β, IL-6, GBP-1, MMP 13, MyD88, BCL2 and
cMyc in a sample from the patient.

DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention provides compounds represented by the
following formula (1):

##STR00002##

and all of their stereoisomeric and tautomeric forms and mixtures
thereof, in all ratios, and their pharmaceutically acceptable salts,
pharmaceutically acceptable solvates, pharmaceutically acceptable
polymorphs and prodrugs, wherein, R1 is selected from halogen,
hydroxy, alkoxy, --O(CO)R13, --SR14, and --NR14R15;
R2 is hydrogen; or optionally R1 is absent and R2 is
═O; R3 is alkyl; R4 is selected from the following
formulae:

##STR00003##

R5 is selected from hydroxy, and alkoxy; R6 is selected from
hydrogen, hydroxy, alkyl, and alkoxy; R7 is selected from hydrogen,
alkyl, and --(CO)R16; R8 is selected from hydroxy, and alkoxy;
R9 is selected from hydroxy, alkyl, alkoxy, aryl, aralkyl, aryloxy,
benzyloxy, heterocyclyl, --O-heterocyclyl, --OCH2COOR17, and
--OCH2COR18; R10 is selected from halogen, hydroxy,
alkoxy, --SR14, --NR14R15, and --O(CO)R19; R11
is selected from hydrogen, and halogen; R12 is selected from
hydrogen, halogen, and hydroxy; R13 is selected from alkyl, and
aryl; R14 is selected from hydrogen, alkyl, aralkyl, aryl, and
heterocyclyl; R15 is selected from hydrogen, and alkyl; R16 is
selected from alkyl, and aryl; R17 is selected from hydrogen, and
alkyl; R18 is selected from alkyl, --NHCH2R20, aryl, and
heterocyclyl; R19 is selected from alkyl, aralkyl, aryl, and
heterocyclyl; and R20 is selected from hydrogen, alkyl, aryl, and
heterocyclyl; where alkyl is unsubstituted or substituted by one or two
of the same or different groups selected from: hydroxy, halogen, amino,
hydroxyalkyl, alkoxy, aryl, aryloxy, and heterocyclyl; alkoxy is
unsubstituted or substituted by one or two of the same or different
groups selected from: halogen, hydroxy, alkyl, and hydroxyalkyl; aryl is
unsubstituted or substituted by one or two of the same or different
groups selected from: halogen, hydroxy, amino, alkyl, hydroxyalkyl,
alkoxy, aryl, and heterocyclyl; heterocyclyl is unsubstituted or
substituted by one or two of the same or different groups selected from:
halogen, hydroxy, amino, alkyl, hydroxyalkyl, alkoxy, aryl, and
heterocyclyl; for the treatment of an inflammatory disorder mediated by
one or more cytokines selected from Tumor Necrosis Factor-alpha
(TNF-α), interferon-γ (IFN-γ) and interleukins such as
IL-113, IL-2, IL-6.

DEFINITIONS

[0028] Listed below are definitions, which apply to the terms as they are
used throughout the specification and the appended claims (unless they
are otherwise limited in specific instances), either individually or as
part of a larger group.

[0029] As used herein, the term "alkyl" whether used alone or as part of a
substituent group, refers to saturated aliphatic groups, including
straight or branched-chain containing from 1 to 6 carbon atoms. Suitable
alkyl groups contain for example, from 1 to 4 carbon atoms, such as
methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, and t-butyl. An
alkyl group is optionally substituted by one or more identical or
different substituents. Any kind of substituent present in substituted
alkyl groups can be present in any desired position provided that the
substitution does not lead to an unstable molecule. A substituted alkyl
refers to an alkyl group in which one or more, for example, 1, 2, 3, 4 or
5 hydrogen atoms are replaced with substituents, for example, halogen,
hydroxy, amino, alkoxy, hydroxyalkyl, aryloxy, acyloxy, aryl, heteroaryl,
or heterocyclyl group.

[0030] As used herein, the term "alkoxy" refers to an alkyl group having
an oxygen attached thereto, wherein alkyl is as defined above.
Representative alkoxy groups include methoxy, ethoxy, propoxy, and
tert-butoxy group. The terms include, therefore, alkoxy groups, which are
substituted by one or more identical or different groups selected from:
halogen, hydroxy, alkyl, and hydroxyalkyl.

[0031] As used herein, the term "aryl" refers to a monocyclic or bicyclic
hydrocarbon group having up to 10 ring carbon atoms, in which at least
one carbocyclic ring is present that has a conjugated π electron
system. Examples of aryl group include phenyl and naphthyl. A substituted
aryl refers to an aryl group, which is substituted by one or more
substituents, for example, up to five identical or different substituents
selected from the group consisting of halogen, hydroxy, amino, alkyl,
hydroxyalkyl, alkoxy, aryloxy, aryl, and a heterocyclyl group. Aryl
groups can be substituted in any desired position. For example, in
monosubstituted phenyl groups, the substituent can be located in the
2-position, the 3-position, the 4-position or the 5-position. If the
phenyl group carries two substituents, they can be located in
2,3-position, 2,4-position, 2,5-position, 2,6-position, 3,4-position or
3,5-position.

[0032] As used herein, the term "aryloxy" refers to the aryl-O-- wherein
the term aryl is as defined above. Exemplary aryloxy groups include, but
are not limited to, phenoxy and naphthoxy. The term "heteroatom" refers
to nitrogen, oxygen and sulfur. It should be noted that any heteroatom
with unsatisfied valences is assumed to have a hydrogen atom to satisfy
the valences. The ring heteroatoms can be present in any desired number
and in any position with respect to each other provided that the
resulting heterocyclic system is stable.

[0033] The terms "heterocyclyl", and "heterocyclic" refer to a saturated,
partially unsaturated or aromatic monocyclic or bicyclic ring system
containing 3, 4, 5, 6, 7, 8, 9, or 10, ring atoms of which 1, 2, 3 or 4
are identical or different heteroatoms selected from: nitrogen, oxygen
and sulfur. The heterocyclyl group may, for example, have 1 or 2 oxygen
atoms and/or 1 or 2 sulfur atoms and/or 1 to 4 nitrogen atoms in the
ring. Heterocyclyl includes saturated heterocyclic ring systems, which do
not contain any double bonds within the rings, as well as unsaturated
heterocyclic ring systems, which contain one or more, up to 5 double
bonds within the rings provided that the resulting system is stable.
Unsaturated rings may be non-aromatic or aromatic. Aromatic heterocyclyl
groups may also be referred to by the customary term "heteroaryl" for
which all the definitions and explanations above and below relating to
heterocyclyl apply. Monocyclic heterocyclyl groups include 3-membered,
4-membered, 5-membered, 6-membered and 7-membered rings. Suitable
examples of such heterocyclyl groups are pyrrolyl, imidazolyl,
pyrrolidinyl, pyridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, pyrazolyl,
triazolyl, tetrazolyl, piperidinyl, piperazinyl, and morpholinyl.
Bicyclic heterocyclyl groups include two fused rings, one of which is 5-,
6- or 7-membered heterocyclic ring and the other of which is a 5-, 6- or
7-membered carbocyclic or heterocyclic ring. Exemplary bicyclic
heterocyclic groups include benzoxazolyl, quinolyl, isoquinolyl, indolyl,
isoindolyl, and benzofurazanyl.

[0034] A substituted heterocyclyl refers to a heterocyclyl group which is
substituted with one or more (up to 5), identical or different
substituents. Examples of substituents for the ring carbon and ring
nitrogen atoms are: halogen, hydroxy, amino, alkyl, hydroxyalkyl, alkoxy,
aryloxy, aryl, and heterocyclyl. The substituents can be present at one
or more positions provided that a stable molecule results.

[0035] As used herein the term "aralkyl" refers to an alkyl group
substituted with an aryl or heteroaryl group, wherein the terms alkyl,
aryl and heteroaryl are as defined above. Exemplary aralkyl groups
include --(CH2)p-phenyl, --(CH2)p-pyridyl, wherein p
is an integer from 1 to 3. The aralkyl group may be further substituted
with hydroxy, halogen, amino, alkyl, aryl or heteroaryl.

[0036] As used herein, the term --O-heterocyclyl refers to the
heterocyclic ring attached directly to an oxygen atom wherein the term
heterocyclyl is as defined above.

[0037] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0038] The term "amino" refers to unsubstituted, mono-substituted and
di-substituted amino groups.

[0039] As used herein, the terms mono- or di-substituted amino refer
respectively to an amino group substituted by one or two groups which may
be the same or different. The substituents on the amino group are
independently selected from: alkyl, hydroxyalkyl, aralkyl, aryl, and
heterocyclyl. It will be understood by those skilled in the art that the
moieties on the amino group can themselves be substituted, if
appropriate.

[0040] The expression "prodrug" refers to compounds that are drug
precursors, which following administration, release the drug in vivo via
a chemical or physiological process e.g., a prodrug on being brought to
the physiological pH or through an enzyme action is converted to the
desired drug form.

[0041] It will be understood that "substitution" or "substituted with"
includes the implicit proviso that such substitution is in accordance
with permitted valence of the substituted atom and the substituent, as
well as results in a stable compound, which does not readily undergo
transformation such as by rearrangement, cyclization, elimination, etc.

[0042] The term "subject" as used herein refers to an animal, preferably a
mammal, and most preferably a human.

[0043] The term "mammal" used herein refers to warm-blooded vertebrate
animals of the class Mammalia, including humans, characterized by a
covering of hair on the skin and, in the female, milk-producing mammary
glands for nourishing the young. The term mammal includes animals such as
cat, dog, rabbit, bear, fox, wolf, monkey, deer, mouse, pig as well as
human.

[0046] The term "treating", "treat" or "treatment" as used herein refers
to alleviate, slow the progression, attenuation or cure of existing
disease (for example, rheumatoid arthritis).

[0047] By "pharmaceutically acceptable" it is meant that the carrier,
diluent, excipients, and/or salt must be compatible with the other
ingredients of the formulation, and not deleterious to the recipient
thereof.

[0048] The term "pharmaceutically acceptable carrier" as used herein means
a non-toxic, inert, solid, semi-solid, diluent, encapsulating material or
formulation auxiliary of any type. Some examples of materials which can
serve as pharmaceutically acceptable carriers are sugars such as lactose,
glucose, and sucrose; starches such as corn starch and potato starch;
cellulose and its derivatives such as sodium carboxymethyl cellulose,
ethyl cellulose and cellulose acetate; malt; gelatin; talc; as well as
other non-toxic compatible lubricants such as sodium lauryl sulfate and
magnesium stearate, as well as coloring agents, releasing agents, coating
agents, sweetening, flavoring and perfuming agents; preservatives and
antioxidants can also be present in the composition, according to the
judgment of the formulator.

[0049] The term, "therapeutically effective amount" as used herein means
an amount of compound or composition (e.g. compound of formula (1))
sufficient to significantly induce a positive modification in the
condition to be regulated or treated, but low enough to avoid undue or
severe side effects, within the scope of sound medical judgment. The
therapeutically effective amount of the compound or composition will vary
with the particular condition being treated, the age and physical
condition of the end user, the severity of the condition being
treated/prevented, the duration of the treatment, the nature of
concurrent therapy, the specific compound or composition employed, the
particular pharmaceutically acceptable carrier utilized, and like
factors. As used herein, all percentages are by weight unless otherwise
specified.

[0050] The term "abnormal" as used herein and in the appended claims in
the context of one or more proinflammatory cytokines selected from Tumor
Necrosis Factor-alpha (TNF-α), interferon-γ (IFN-γ) and
interleukins such as IL-1β, IL-2, IL-6 and IL-8 refers to elevated
or increased levels of the proinflammatory cytokines.

EMBODIMENTS OF THE INVENTION

[0051] The present invention provides compounds represented by the
following formula (1),

##STR00004##

and all of their stereoisomeric and tautomeric forms and mixtures
thereof, in all ratios, and their pharmaceutically acceptable salts,
pharmaceutically acceptable solvates, pharmaceutically acceptable
polymorphs and prodrugs, wherein, R1 is selected from halogen,
hydroxy, alkoxy, --O(CO)R13, --SR14, and --NR14R15;
R2 is hydrogen; R3 is alkyl; R4 is selected from the
following formulae:

##STR00005##

R5 is selected from hydroxy, and alkoxy; R6 is selected from
hydrogen, hydroxy, alkyl, and alkoxy; R7 is selected from hydrogen,
alkyl, and --(CO)R16; R8 is selected from hydroxy, and alkoxy;
R9 is selected from hydroxy, alkyl, alkoxy, aryl, aralkyl, aryloxy,
benzyloxy, heterocyclyl, --O-heterocyclyl, --OCH2COOR17, and
--OCH2COR18; R10 is selected from halogen, hydroxy,
alkoxy, --SR14, --NR14R15, and --O(CO)R19; R11
is selected from hydrogen, and halogen; R12 is selected from
hydrogen, halogen, and hydroxy; R13 is selected from alkyl, and
aryl; R14 is selected from hydrogen, alkyl, aralkyl, aryl, and
heterocyclyl; R15 is selected from hydrogen, and alkyl; R16 is
selected from alkyl, and aryl; R17 is selected from hydrogen, and
alkyl; R18 is selected from alkyl, --NHCH2R20, aryl, and
heterocyclyl; R19 is selected from alkyl, aralkyl, aryl, and
heterocyclyl; and R20 is selected from hydrogen, alkyl, aryl, and
heterocyclyl; where alkyl is unsubstituted or substituted by one or two
of the same or different groups selected from: hydroxy, halogen, amino,
hydroxyalkyl, alkoxy, aryl, aryloxy, and heterocyclyl; alkoxy is
unsubstituted or substituted by one or two of the same or different
groups selected from: halogen, hydroxy, alkyl, and hydroxyalkyl; aryl is
unsubstituted or substituted by one or two of the same or different
groups selected from: halogen, hydroxy, amino, alkyl, hydroxyalkyl,
alkoxy, aryl, and heterocyclyl; heterocyclyl is unsubstituted or
substituted by one or two of the same or different groups selected from:
halogen, hydroxy, amino, alkyl, hydroxyalkyl, alkoxy, aryl, and
heterocyclyl; for the treatment of an inflammatory disorder mediated by
one or more cytokines selected from Tumor Necrosis Factor-alpha
(TNF-α), interferon-γ (IFN-γ) and interleukins such as
IL-1(i, IL-6, and IL-8.

R1 is selected from halogen, hydroxy, and alkoxy; R2 is
hydrogen; R3 is alkyl; R4 is formula (3):

##STR00006##

R8 is hydroxy; R9 is selected from hydroxy, alkyl, alkoxy,
aryl, aralkyl, aryloxy, benzyloxy, --OCH2COOR17, and
--OCH2COR18; R17 is selected from hydrogen, and alkyl;
R18 is selected from alkyl, --NHCH2R20, aryl, and
heterocyclyl; and R20 is selected from hydrogen, alkyl, aryl, and
heterocyclyl; where alkyl is unsubstituted or substituted by one or two
of the same or different groups selected from: hydroxy, halogen, amino,
hydroxyalkyl, alkoxy, aryl, aryloxy, and heterocyclyl; alkoxy is
unsubstituted or substituted by one or two of the same or different
groups selected from: halogen, hydroxy, alkyl, and hydroxyalkyl; aryl is
unsubstituted or substituted by one or two of the same or different
groups selected from: halogen, hydroxy, amino, alkyl, hydroxyalkyl,
alkoxy, aryl, and heterocyclyl; heterocyclyl is unsubstituted or
substituted by one or two of the same or different groups selected from:
halogen, hydroxy, amino, alkyl, hydroxyalkyl, alkoxy, aryl, and
heterocyclyl.

[0053] In a further embodiment, the present invention provides compounds
represented by formula (1), wherein,

R1 is hydroxy; R2 is hydrogen; R3 is alkyl; R4 is
formula (3):

##STR00007##

R8 is hydroxy; and R9 is selected from hydroxy, alkyl, alkoxy,
and benzyloxy; where alkyl is unsubstituted or substituted by one or two
of the same or different groups selected from: hydroxy, halogen, amino,
hydroxyalkyl, and alkoxy.

[0054] In a further embodiment, the present invention provides compounds
represented by formula (1), wherein,

R1 is hydroxy; R2 is hydrogen; R3 is methyl; R4 is
formula (3):

##STR00008##

R8 is hydroxy; and R9 is selected from hydroxy, methoxy, and
benzyloxy.

[0055] In a further embodiment, the present invention provides compound
represented by formula (1), wherein,

R1 is selected from halogen, hydroxy, and alkoxy; R2 is
hydrogen; R3 is alkyl; R4 is formula (3):

##STR00010##

R8 is selected from hydroxy, and alkoxy; R9 is selected from
--OCH2COOR17, and --OCH2COR18; R17 is selected
from hydrogen, and alkyl; R18 is selected from alkyl, heterocyclyl
and --NHCH2R20; and R20 is selected from hydrogen, alkyl,
aryl, and heterocyclyl; where alkyl is unsubstituted or substituted by
one or two of the same or different groups selected from: hydroxy,
halogen, amino, hydroxyalkyl, and alkoxy; alkoxy is unsubstituted or
substituted by one or two of the same or different groups selected from:
hydroxy, alkyl, and hydroxyalkyl; aryl is unsubstituted or substituted by
one or two of the same or different groups selected from: halogen,
hydroxy, hydroxyalkyl, alkoxy, aryl, and heterocyclyl; heterocyclyl is
unsubstituted or substituted by one or two of the same or different
groups selected from: hydroxy, hydroxyalkyl, alkyl, alkoxy, aryl, and
heterocyclyl.

[0057] In a further embodiment, the present invention provides compounds
represented by formula (1), wherein,

[0062] R20 is selected from alkyl, and aryl; where alkyl is
unsubstituted or substituted by one or two of the same or different
groups selected from: hydroxy, halogen, amino, hydroxyalkyl, and alkoxy;
aryl is unsubstituted or substituted by one or two of the same or
different groups selected from: halogen, hydroxy, and alkoxy.

R1 is selected from halogen, hydroxy, and alkoxy; R2 is
hydrogen; R3 is alkyl; R4 is formula (6):

##STR00015##

R10 is selected from halogen, hydroxy, and alkoxy; where alkyl is
unsubstituted or substituted by one or two of the same or different
groups selected from: hydroxy, halogen, amino, hydroxyalkyl, and alkoxy;
alkoxy is unsubstituted or substituted by one or two of the same or
different groups selected from: halogen, hydroxy, alkyl, and
hydroxyalkyl.

R1 is selected from halogen, hydroxy, and alkoxy; R2 is
hydrogen; R3 is alkyl; R4 is formula (2):

##STR00020##

R5 is selected from hydroxy, and alkoxy; R6 is selected from
hydrogen, alkyl, hydroxy, and alkoxy; R7 is selected from hydrogen,
alkyl, and --(CO)R16; and R16 is selected from alkyl, and aryl;
where alkyl is unsubstituted or substituted by one or two of the same or
different groups selected from: hydroxy, halogen, amino, hydroxyalkyl,
and alkoxy; alkoxy is unsubstituted or substituted by one or two of the
same or different groups selected from: halogen, hydroxy, alkyl, and
hydroxyalkyl; aryl is unsubstituted or substituted by one or two of the
same or different groups selected from: halogen, hydroxy, amino, alkyl,
hydroxyalkyl, alkoxy, aryl, and heterocyclyl.

R1 is selected from halogen, hydroxy, and alkoxy; R2 is
hydrogen; R3 is alkyl; R4 is formula (2):

##STR00021##

R5 is selected from hydroxy, and alkoxy; R6 is selected from
hydrogen, and hydroxy; R7 is selected from hydrogen, alkyl, and
--(CO)R16; and R16 is alkyl; where alkyl is unsubstituted or
substituted by one or two of the same or different groups selected from:
hydroxy, halogen, amino, hydroxyalkyl, and alkoxy; alkoxy is
unsubstituted or substituted by one or two of the same or different
groups selected from: halogen, hydroxy, alkyl, and hydroxyalkyl.

R10 is selected from halogen, hydroxy, alkoxy, --SR14,
--NR14R15, and --O(CO)R19; R13 is selected from
alkyl, and aryl; R14 is selected from hydrogen, alkyl, aralkyl,
aryl, and heterocyclyl; R15 is selected from hydrogen, and alkyl;
and R19 is selected from alkyl, aryl, and heterocyclyl; where alkyl
is unsubstituted or substituted by one or two of the same or different
groups selected from: hydroxy, halogen, amino, hydroxyalkyl, alkoxy,
aryl, aryloxy, and heterocyclyl; alkoxy is unsubstituted or substituted
by one or two of the same or different groups selected from: halogen,
hydroxy, alkyl, and hydroxyalkyl; aryl is unsubstituted or substituted by
one or two of the same or different groups selected from: halogen,
hydroxy, amino, alkyl, hydroxyalkyl, alkoxy, aryl, and heterocyclyl;
heterocyclyl is unsubstituted or substituted by one or two of the same or
different groups selected from: halogen, hydroxy, amino, alkyl,
hydroxyalkyl, alkoxy, aryl, and heterocyclyl.

[0076] R14 is selected from hydrogen, and alkyl; where alkyl is
unsubstituted or substituted by one or two of the same or different
groups selected from: hydroxy, halogen, amino, hydroxyalkyl, and alkoxy.

R10 is selected from halogen, hydroxy, alkoxy, --SR14,
--NR14R15, and --O(CO)R19; R14 is selected from
hydrogen, alkyl, aralkyl, aryl, and heterocyclyl; R15 is selected
from hydrogen, and alkyl; and R19 is selected from alkyl, aryl, and
heterocyclyl; where alkyl is unsubstituted or substituted by one or two
of the same or different groups selected from: hydroxy, halogen, amino,
hydroxyalkyl, alkoxy, aryl, aryloxy, and heterocyclyl; alkoxy is
unsubstituted or substituted by one or two of the same or different
groups selected from: halogen, hydroxy, alkyl, and hydroxyalkyl; aryl is
unsubstituted or substituted by one or two of the same or different
groups selected from: halogen, hydroxy, amino, alkyl, hydroxyalkyl, and
alkoxy; heterocyclyl is unsubstituted or substituted by one or two of the
same or different groups selected from: halogen, hydroxy, amino, alkyl,
hydroxyalkyl, and alkoxy.

[0081] R14 is selected from hydrogen, and alkyl; and R15 is
selected from hydrogen, and alkyl; where alkyl is unsubstituted or
substituted by one or two of the same or different groups selected from:
hydroxy, halogen, amino, hydroxyalkyl, and alkoxy;

[0082] In one embodiment, the present invention relates to the use of a
compound of formula (1), wherein,

[0086] R13 is selected from alkyl, and aryl; and R19 is selected
from alkyl, aralkyl, aryl, and heterocyclyl; where alkyl is unsubstituted
or substituted by one or two of the same or different groups selected
from: hydroxy, halogen, amino, hydroxyalkyl, and alkoxy; aryl is
unsubstituted or substituted by one or two of the same or different
groups selected from: halogen, hydroxy, amino, alkyl, hydroxyalkyl, and
alkoxy; heterocyclyl is unsubstituted or substituted by one or two of the
same or different groups selected from: halogen, hydroxy, amino, alkyl,
hydroxyalkyl, and alkoxy.

R10 is hydroxy; and R13 is selected from alkyl, and aryl; where
alkyl is unsubstituted or substituted by one or two of the same or
different groups selected from: hydroxy, halogen, amino, hydroxyalkyl,
and alkoxy; aryl is unsubstituted or substituted by one or two of the
same or different groups selected from: halogen, hydroxy, amino, alkyl,
hydroxyalkyl, and alkoxy.

R10 is selected from halogen, hydroxy, alkoxy, --SR14,
--NR14R15 and --O(CO)R19; R14 is selected from
hydrogen, alkyl, aralkyl, aryl, and heterocyclyl; R15 is selected
from hydrogen, and alkyl; and R19 is selected from alkyl, and aryl;
where alkyl is unsubstituted or substituted by one or two of the same or
different groups selected from: hydroxy, halogen, amino, hydroxyalkyl,
alkoxy, aryl, aryloxy, and heterocyclyl; alkoxy is unsubstituted or
substituted by one or two of the same or different groups selected from:
halogen, hydroxy, alkyl, and hydroxyalkyl; aryl is unsubstituted or
substituted by one or two of the same or different groups selected from:
halogen, hydroxy, amino, alkyl, hydroxyalkyl, and alkoxy; heterocyclyl is
unsubstituted or substituted by one or two of the same or different
groups selected from: halogen, hydroxy, amino, alkyl, hydroxyalkyl, and
alkoxy.

R10 is selected from halogen, hydroxy, alkoxy, --SR14,
--NR14R15, and --O(CO)R19; R14 is selected from
hydrogen, alkyl, aralkyl, aryl, and heterocyclyl; R15 is selected
from hydrogen, and alkyl; and R19 is selected from alkyl, aryl,
aralkyl, and heterocyclyl; where alkyl is unsubstituted or substituted by
one or two of the same or different groups selected from: hydroxy,
halogen, amino, hydroxyalkyl, and alkoxy; alkoxy is unsubstituted or
substituted by one or two of the same or different groups selected from:
halogen, hydroxy, alkyl, and hydroxyalkyl; aryl is unsubstituted or
substituted by one or two of the same or different groups selected from:
halogen, hydroxy, amino, alkyl, hydroxyalkyl, and alkoxy; heterocyclyl is
unsubstituted or substituted by one or two of the same or different
groups selected from: halogen, hydroxy, amino, alkyl, hydroxyalkyl, and
alkoxy.

R1 is selected from halogen, hydroxy, and alkoxy; R2 is
hydrogen; R3 is alkyl; R4 is formula (8):

##STR00036##

R10 is selected from halogen, and hydroxy; where alkyl is
unsubstituted or substituted by one or two of the same or different
groups selected from: hydroxy, halogen, amino, hydroxyalkyl, alkoxy,
aryl, aryloxy, and heterocyclyl; alkoxy is unsubstituted or substituted
by one or two of the same or different groups selected from: halogen,
hydroxy, alkyl, and hydroxyalkyl.

R1 is selected from halogen, hydroxy, and alkoxy; R2 is
hydrogen; R3 is alkyl; and R4 is formula (9):

##STR00038##

where alkyl is unsubstituted or substituted by one or two of the same or
different groups selected from: hydroxy, halogen, amino, hydroxyalkyl,
alkoxy, aryl, aryloxy, and heterocyclyl; alkoxy is unsubstituted or
substituted by one or two of the same or different groups selected from:
halogen, hydroxy, alkyl, and hydroxyalkyl.

[0097] The present invention provides compounds of formula (1) (as
provided in the above given all embodiments), and all of their
stereoisomeric and tautomeric forms and mixtures thereof, in all ratios,
and their pharmaceutically acceptable salts, pharmaceutically acceptable
solvates, pharmaceutically acceptable polymorphs and prodrugs, for the
treatment of an inflammatory disorder mediated by one or more cytokines
selected from Tumor Necrosis Factor-alpha (TNF-α),
interferon-γ (IFN-γ) and interleukins such as IL-1β,
IL-2, IL-6, and IL-8.

[0098] The present invention provides compounds of formula (1) (as
provided in the above given all embodiments), and all of their
stereoisomeric and tautomeric forms and mixtures thereof, in all ratios,
and their pharmaceutically acceptable salts, pharmaceutically acceptable
solvates, pharmaceutically acceptable polymorphs and prodrugs, for the
treatment of an inflammatory disorder mediated by one or more genes
selected from CEBPα, CEBPβ, CEBPδ, IL-1β, IL-6,
GBP-1, MMP 13, MyD88, BCL2 and cMyc.

[0099] The present invention relates to the use of a compound of formula
(1) is selected from but not limited to:

##STR00039## ##STR00040## ##STR00041##

their stereoisomeric and tautomeric forms, pharmaceutically acceptable
salts, solvates and prodrugs; for the treatment of an inflammatory
disorder mediated by one or more cytokines selected from Tumor Necrosis
Factor-alpha (TNF-α), interferon-γ (IFN-γ) and
interleukins such as IL-1(3, IL-2, IL-6, and IL-8.

[0100] In another further aspect of the invention, the compound of formula
(1) is selected from:

##STR00042## ##STR00043##

their stereoisomeric and tautomeric forms, pharmaceutically acceptable
salts, solvates and prodrugs; for the treatment of an inflammatory
disorder mediated by one or more cytokines selected from Tumor Necrosis
Factor-alpha (TNF-α), interferon-γ (IFN-γ) and
interleukins such as IL-1β, IL-2, IL-6, and IL-8.

[0101] In yet another further aspect of the invention, the compound of
formula (1) is:

##STR00044##

its stereoisomeric and tautomeric forms, pharmaceutically acceptable
salts, solvates and prodrugs; for the treatment of an inflammatory
disorder mediated by one or more cytokines selected from Tumor Necrosis
Factor-alpha (TNF-α), interferon-γ (IFN-γ) and
interleukins such as IL-1β, IL-2, IL-6, and IL-8.

DETAILED DESCRIPTION OF THE SCHEMES

[0102] The compounds of the present invention also include all
stereoisomeric forms and mixtures thereof and their pharmaceutically
acceptable salts, solvates and polymorphs. Furthermore, all prodrugs and
derivatives of the compounds are a subject of the present invention.

[0103] According to another aspect of present invention, the compounds of
formula (1) can be prepared in a number of ways including using methods
well known to the person skilled in the art. Examples of methods to
prepare the present compounds are described below and illustrated in
Schemes 1 to 4 but are not limited thereto. It will be appreciated by
persons skilled in the art that the processes described herein, the order
of the synthetic steps employed may be varied and will depend inter alia
on factors such as the nature of functional groups present in a
particular substrate and the protecting group strategy (if any) to be
adopted and will also influence the choice of reagent to be used in the
synthetic steps.

[0104] The reagents, reactants and intermediates used in the following
processes are either isolated from fermentation of microorganisms, are
commercially available or can be prepared according to standard
literature procedures known in the art or a combination thereof. The
starting compounds and the intermediates used for the synthesis of
compounds of the present invention, are referred to with general symbols
namely (A), (B), (C), (D), (E), (F), (G), (H), (K), (L), (M), (N), (O),
(O), (R), (S), (T), and (U). Throughout the process description, the
corresponding substituent groups in the various formulae representing
starting compounds and intermediates have the same meanings as that for
the compounds of formula (1) as described in detailed description.

[0105] The processes used in various schemes of the present invention, are
referred to with general symbols namely 1a, 1b, 1c, 1d, 1e, 1f, 1 g, 2a,
2b, 2c, 2d, 3a, 3b, 3c, 4a, 4b, 4c, and 4d. Processes for the preparation
of compounds of the present invention are set forth in the following
schemes:

Scheme 1

[0106] Concanamycin crude (in Scheme 1) is obtained by fermentation of a
culture (PM0224355). The whole broth is extracted using a solvent
selected from ethyl acetate, chloroform and dichloromethane. Concanamycin
crude is isolated by column chromatography and is characterized by
spectral comparison (The Journal of Antibiotics, Vol. 45, No. 7,
1108-1116, (1992)).

[0108] Compound of formula (1) (wherein R1 is hydroxy, R2 is
hydrogen, R3 is methyl, R4 is formula (3), R8 is hydroxy,
and R9 is hydroxy; denoted as formula (B) in Scheme 1) is prepared
by reacting compound of formula (A) (wherein R1 is hydroxy, R2
is hydrogen, R3 is methyl, R4 is formula (4), and R10 is
hydroxy) with an amine hydrochloride such as hydroxylamine hydrochloride
in presence of a base selected from pyridine, substituted pyridine,
triethylamine, diisopropylethylamine, N-methylmorpholine, and
N-ethylmorpholine using a solvent selected from methanol, ethanol,
propanol, butanol, tetrahydrofuran, dimethylformamide, 1,4-dioxane, and
acetonitrile. The reaction mixture is stirred at a temperature in the
range of 0° C. to 45° C. in an inert atmosphere such as
nitrogen gas, over a time period ranging from 4 h to 16 h.

Step 1c

[0109] Compound of formula (1) (wherein R1 is hydroxy, R2 is
hydrogen, R3 is methyl, R4 is formula (6), and R10 is
hydroxy; denoted as formula (C) in Scheme 1) is prepared by reacting
compound of formula (B) (wherein R1 is hydroxy, R2 is hydrogen,
R3 is methyl, R4 is formula (3), R8 is hydroxy, and
R9 is hydroxy) in a solvent selected from acetone, acetonitrile, and
1,4-dioxane with tosyl chloride or 2,4,6-trichloro-1,3,5-triazine (TCT),
in presence of a base selected from sodium hydroxide and potassium
hydroxide, in an inert atmosphere such as nitrogen at 0° C., for 2
h. The reaction mixture can be further stirred at a temperature in the
range of 25° C. to 45° C., in an inert atmosphere such as
nitrogen gas, over a time period ranging from 2 h to 8 h.

Step 1d

[0110] Compound of formula (1) (wherein R1 is hydroxy, R2 is
hydrogen, R3 is methyl, R4 is formula (3), R8 is hydroxy,
and R9 is methoxy or benzyloxy; denoted as formula (D) in Scheme 1)
is prepared by reacting compound of formula (A) (wherein R1 is
hydroxy, R2 is hydrogen, R3 is methyl, R4 is formula (4),
and R10 is hydroxy) with an amine hydrochloride selected from
methoxyamine hydrochloride, and benzyloxy amine hydrochloride in presence
of a base selected from pyridine, substituted pyridine, triethylamine,
diisopropylethylamine, N-methylmorpholine, and N-ethylmorpholine using a
solvent selected from methanol, ethanol, propanol, butanol,
tetrahydrofuran, dimethylformamide, dioxane, and acetonitrile. The
reaction mixture is stirred at a temperature in the range of 0° C.
to 45° C., in an inert atmosphere such as nitrogen gas, over a
time period ranging from 4 h to 16 h.

Step 1e

[0111] Compound of formula (1) (wherein R1 is hydroxy, R2 is
hydrogen and R3 is methyl, R4 is formula (3), R8 is
hydroxy, R9 is --OCH2COOR17, and R17 is hydrogen;
denoted as formula (E) in Scheme1) is prepared by reacting compound of
formula (A) (wherein R1 is hydroxy, R2 is hydrogen, R3 is
methyl, R4 is formula (4), and R10 is hydroxy) with amine
hydrochloride such as carboxymethylhydroxylamine hemi hydrochloride in
presence of a base selected from pyridine, substituted pyridine,
triethylamine, diisopropylethylamine, N-methylmorpholine and
N-ethylmorpholine using a solvent selected from methanol, ethanol,
propanol, butanol, tetrahydrofuran, dimethylformamide, 1,4-dioxane, and
acetonitrile. The reaction mixture is stirred at a temperature in the
range of 0° C. to 45° C., in an inert atmosphere such as
nitrogen gas, over a time period ranging from 4 h to 16 h.

Step 1f

[0112] Compound of formula (1) (wherein R1 is hydroxy, R2 is
hydrogen, R3 is methyl, R4 is formula (3), R8 is hydroxy,
R9 is --OCH2COR18, R18 is selected from heterocyclyl
and --NHCH2R20, and R20 is selected from alkyl, and aryl;
denoted as formula (F) in Scheme 1) is prepared by dissolving compound of
formula (E) (wherein R1 is hydroxy, R2 is hydrogen, R3 is
methyl, R4 is formula (3), R8 is hydroxy, R9 is
--OCH2COOR17, and R17 is hydrogen) in a solvent selected
from dichloromethane, acetonitrile, chloroform, ethyl acetate, and
dimethylformamide, and reacting with a coupling reagent selected from
dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride

[0113] (EDC HCl), N,N'-diisopropyl carbodi imide (DIC), or
O-benzotriazol-1-yl-N,N,N',N'-tetramethyl uranium hexa fluorophosphate
(HBTU),O-benzotriazol-1-yl-N,N,N',N'-tetramethyluroniumtetrafluoroborate(-
TBTU), benzotriazol-1-yl-oxytripyrrolidinophosphonium
hexafluorophosphate(PyBOP) and N-hydroxy benzotriazole (HOBt). Further,
the reaction mixture is treated with an amine such as
N-methyl-piperazine, ethanolamine, piperidine, 4-piperidino-piperidine,
and 4-fluoro phenylamine. The reaction mixture is stirred at a
temperature in the range of 25° C. to 45° C., in an inert
atmosphere such as nitrogen gas, over a time period ranging from 4 h to
18 h.

Step 1 g

[0114] Compound of formula (1) (wherein R1 is hydroxy, R2 is
hydrogen, R3 is methyl, R4 is formula (7), R10 is hydroxy,
R11 is hydrogen, and R12 is hydroxy; denoted as formula (G) in
Scheme 1) is prepared by dissolving compound of formula (A) (wherein
R1 is hydroxy, R2 is hydrogen, R3 is methyl, R4 is
formula (4), and R10 is hydroxy) in a solvent selected from
tetrahydrofuran, acetonitrile, acetone, methanol and ethanol, and is
reacted with a reducing agent such as sodium borohydride, in an inert
atmosphere such as nitrogen at 0° C. for 20 min. The reaction
mixture is further stirred at a temperature in the range of 25° C.
to 45° C., in an inert atmosphere such as nitrogen gas, over a
time period ranging from 2 h to 8 h.

##STR00045##

Step 2a

[0115] Compound of formula (1) (wherein R1 is hydroxy, R2 is
hydrogen, R3 is methyl, R4 is formula (8), and R10 is
hydroxy; denoted as formula (H), in Scheme 2) is prepared by dissolving
compound of formula (B) (wherein R1 is hydroxy, R2 is hydrogen,
R3 is methyl; R4 is formula (3), R8 is hydroxy, and
R9 is hydroxy; prepared by step 1b, Scheme 1) in a solvent selected
from acetone, acetonitrile, 1,4-dioxane, with reagent such as tosyl
chloride, in presence of a base selected from sodium hydroxide and
potassium hydroxide, in an inert atmosphere such as nitrogen at 0°
C., for 2 h. The reaction mixture can be further stirred at a temperature
in the range of 25° C. to 45° C., in an inert atmosphere
such as nitrogen gas, over a time period ranging from 2 h to 8 h.

Step 2b

[0116] Compound of formula (1) (wherein R1 is hydroxy, R2 is
hydrogen, R3 is methyl; and R4 is formula (9); denoted as
formula (K) in Scheme 2) is prepared by dissolving compound of formula
(E) (wherein R1 is hydroxy, R2 is hydrogen, R3 is methyl,
R4 is formula (3), R8 is hydroxy and R9 is
--OCH2COOR17, R17 is hydrogen; prepared by step 1e, Scheme
1)) in a solvent selected from dichloromethane, acetonitrile, chloroform,
ethyl acetate, and dimethylformamide, and reacting with coupling reagent
selected from dicyclohexylcarbodiimide,
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC HCl) and
N,N'-diisopropyl carbodiimide (DIC) and catalyst such as
4-dimethylaminopyridine (DMAP). The reaction mixture is stirred at a
temperature in the range of 25° C. to 45° C., in an inert
atmosphere such as nitrogen gas, over a time period ranging from 4 h to
18 h.

Step 2c

[0117] Compound of formula (1) (wherein R1 is absent, R2 is
═O, R3 is methyl, and R4 is formula (4), and R10 is
hydroxy; denoted as formula (L), in Scheme 2) is prepared by reacting
compound of formula (A) (wherein R1 is hydroxy, R2 is hydrogen,
R3 is methyl, R4 is formula (4), and R10 is hydroxy) in a
solvent selected from dichloromethane, diethyl ether, and tetrahydrofuran
with an oxidizing agent selected from Dess-martin periodinane, pyridinium
dichromate, pyridinium chlorochromate, and Swern oxidizing agent in an
inert atmosphere such as nitrogen at 0° C. for 2 h. The reaction
mixture is further stirred at a temperature in the range of 25° C.
to 45° C., under inert atmosphere such as nitrogen gas, over a
time period ranging from 2 h to 8 h.

Step 2d

[0118] Compound of formula (1) (wherein R1 is absent, R2 is
═O, R3 is methyl, and R4 is formula (5); denoted as formula
(M), in Scheme 2) is prepared by reacting compound of formula (A)
(wherein R1 is hydroxy, R2 is hydrogen, R3 is methyl,
R4 is formula (4) and R10 is hydroxy) in a solvent selected
from dichloromethane, diethyl ether, and tetrahydrofuran with an
oxidizing agent selected from Dess-martin periodinane, pyridinium
dichromate, pyridinium chlorochromate, and Swern oxidizing agent in an
inert atmosphere such as nitrogen at 0° C. for 2 h. The reaction
mixture is further stirred at a temperature in the range of 25° C.
to 45° C., under inert atmosphere such as nitrogen gas, over a
time period ranging from 2 h to 8 h.

##STR00046##

Scheme 3

Step 3a

[0119] Compound of formula (1) (wherein R1 is hydroxy, R2 is
hydrogen, R3 is methyl, R4 is formula (4), and R10 is
--OC(O)R19, R19 is selected from alkyl, aralkyl, aryl, and
heterocyclyl; denoted as formula (N), in Scheme 3) is prepared by
dissolving compound of formula (A) (wherein R1 is hydroxy, R2
is hydrogen, R3 is methyl, R4 is formula (4) and R10 is
hydroxy) in a solvent selected from dichloromethane, acetonitrile,
chloroform, ethyl acetate, and dimethylformamide, and reacting with a
coupling reagent selected from dicyclohexylcarbodiimide or EDC HCl or DIC
in presence of a catalyst such as DMAP. Further, the reaction mixture is
treated with R19--COOH(R19 is selected from alkyl, aralkyl,
aryl, and heterocyclyl) is added to the reaction mixture and at a
temperature in the range of 25° C. to 45° C., in an inert
atmosphere such as nitrogen gas, over a time period ranging from 4 h to
18 h.

Step 3b

[0120] Compound of formula (1) (wherein R1 is hydroxy, R2 is
hydrogen, R3 is selected from ethyl, n-propyl, n-butyl and n-pentyl;
R4 is formula (4), and R10 is hydroxy; denoted as formula (O),
in Scheme 3) is prepared by dissolving a compound of formula (A) (wherein
R1 is hydroxy, R2 is hydrogen, R3 is methyl, R4 is
formula (3), and R10 is hydroxy), in a solvent selected from
dichloromethane, acetonitrile, chloroform, ethyl acetate, and
dimethylformamide, and reacting with R3--OH (wherein R3 is
selected from ethyl, n-propyl, n-butyl and n-pentyl) in presence of
para-toluene sulphonic acid. The reaction mixture is stirred at a
temperature in the range of 25° C. to 45° C., under inert
atmosphere such as nitrogen gas, over a time period ranging from 4 h to
18 h.

Step 3c

[0121] Compound of formula (1) (wherein R1 is hydroxy, R2 is
hydrogen, R3 is methyl, R4 is formula (2), R5 is hydroxy,
R6 is hydrogen, and R7 is selected from hydrogen or alkyl;
denoted as formula (Q), in Scheme 3) is prepared by dissolving a compound
of formula (B) (wherein R1 is hydroxy, R2 is hydrogen, R3
is methyl, R4 is formula (3), R8 is hydroxy and R9 is
hydroxy; prepared by step 1b, Scheme 1) in a solvent selected from
dichloromethane, acetonitrile, chloroform, ethyl acetate, and
dimethylformamide, and is reacted with R7-halide (wherein R7 is
hydrogen or alkyl) in presence of a base selected from triethylamine,
diisopropylethylamine, N-methylmorpholine, and N-ethylmorpholine. The
reaction mixture is stirred at a temperature in the range of 25°
C. to 45° C., in an inert atmosphere such as nitrogen gas, over a
time period ranging from 4 h to 18 h.

##STR00047##

Scheme 4

Step 4a

[0122] Compound of formula (1) (wherein R1 is hydroxy, R2 is
hydrogen, R3 is methyl, R4 is formula (7), R10 is hydroxy,
R11 is halogen and R12 is halogen; denoted as formula (R), in
Scheme 4) is prepared by reacting compound of formula (A) (wherein
R1 is hydroxy, R2 is hydrogen, R3 is alkyl, R4 is
formula (4), and R10 is hydroxy) with an halogenating agent such as
diethylaminosulfur trifluoride (DAST) in a solvent selected from
tetrahydrofuran, dimethylformamide, 1,4-dioxane and acetonitrile at a
temperature in the range of 0° C. to 45° C., in an inert
atmosphere such as nitrogen gas, over a time period ranging from 2 h to 8
h.

Step 4b

[0123] Compound of formula (1) (wherein R1 is halogen, R2 is
hydrogen, R3 is methyl, R4 is formula (4), and R10 is
halogen; denoted as formula (S), in Scheme 4) is prepared by reacting
compound of formula (A) (wherein R1 is hydroxy, R2 is hydrogen,
R3 is methyl, R4 is formula (4), and R10 is hydroxy) with
an halogenating agent such as diethylaminosulfur trifluoride (DAST) in a
solvent selected from tetrahydrofuran, dimethylformamide, 1,4-dioxane and
acetonitrile at a temperature in the range of 0° C. to 45°
C., in an inert atmosphere such as nitrogen gas, over a time period
ranging from 2 h to 8 h.

Step 4c

[0124] Compound of formula (1) (wherein R1 is amino, R2 is
hydrogen, R3 is methyl, R4 is formula (4), R10 is
--NR14R15, R14 is selected from alkyl, aralkyl, aryl and
heterocyclyl and R15 is selected from hydrogen and alkyl; denoted as
formula (T), in Scheme 4) is prepared by reacting compound of formula (A)
(wherein R1 is hydroxy, R2 is hydrogen, R3 is methyl,
R4 is formula (4), and R10 is hydroxy) with sodium
triacetoxyborohydride or sodium cyanoborohydride and amine selected from
R14--NH2 and R14--NH-alkyl (wherein R14 is selected
from alkyl, aralkyl, aryl, and heterocyclyl) in a solvent selected from
benzene, toluene, tetrahydrofuran, dimethylformamide, 1,4-dioxane and
acetonitrile at a temperature in the range of 0° C. to 45°
C., in an inert atmosphere such as nitrogen gas, over a time period
ranging from 2 h to 8 h.

Step 4d

[0125] Compound of formula (1) (wherein R1 is SH, R2 is
hydrogen, R3 is methyl, R4 is formula (4), and R10 is
--SR14, R14 is selected from alkyl, aralkyl, aryl and
heterocyclyl; denoted as formula (U), in Scheme 4) is prepared by
reacting compound of formula (A) (wherein R1 is hydroxy, R2 is
hydrogen, R3 is methyl, R4 is formula (4), and R10 is
hydroxy) with a reducing agent selected from sodium triacetoxyborohydride
and sodium cyanoborohydride and R14--SH (wherein R14 is
selected from alkyl, aralkyl, aryl and heterocyclyl) in presence of a
solvent selected from solvent benzene or toluene, tetrahydrofuran,
dimethylformamide, 1,4-dioxane and acetonitrile at a temperature in the
range of 0° C. to 45° C., in an inert atmosphere such as
nitrogen gas, over a time period ranging from 2 h to 8 h.

##STR00048##

[0126] In all the above mentioned schemes 1 to 4, wherever applicable the
compounds may be optionally converted into their prodrugs and salts.
Additionally the compounds can be separated into individual isomers by
techniques well known in the art such as column chromatography.

[0127] It will be appreciated by those skilled in the art that the
compounds of the present invention can also be utilized in the form of
their pharmaceutically acceptable salts or solvates thereof.

[0128] With respect to the compounds of formula (1) the present invention
also includes all stereoisomeric forms and mixtures thereof in all ratios
and their pharmaceutically acceptable salts.

[0129] The compounds of the present invention can subsequently be
converted into their organic or inorganic salts.

[0131] The compounds of the present invention represented by the formula
(1) contain one or more acidic group they can form an addition salt with
a suitable base. For example, such salts of the compounds of the present
invention may include their alkali metal salts such as Li, Na, and K
salts, or alkaline earth metal salts like Ca, Mg salts, or aluminium
salts, or salts with ammonia or salts of organic bases such as lysine,
arginine, guanidine, diethanolamine, choline, and tromethamine.

[0132] The present invention furthermore includes solvates of the
compounds of formula (1), for example hydrates with water and the
solvates formed with other solvents of crystallization, such as alcohols,
ethers, ethyl acetate, dioxane, dimethylformamide or a lower alkyl ketone
such as acetone, or mixtures thereof.

[0133] The present invention furthermore includes polymorphs of the
compounds of formula (1). Polymorphs may be obtained by heating or
melting the compounds of present invention followed by gradual or fast
cooling. The presence of polymorphs may be determined by techniques such
as IR spectroscopy, solid probe NMR spectroscopy, differential scanning
calorimetry, or powder X-ray diffraction.

[0134] The present invention also includes prodrugs of the compounds of
formula (1), for example esters, amides and other derivatives.

[0136] In certain embodiments, compounds of the invention represented by
formula (1), are interleukin (IL-1β, IL-2, IL-6 and IL-8) inhibitors
and find use in therapies for disorders associated with abnormal
interleukin (IL-1β, IL-2, IL-6 and IL-8) activity, including:
rheumatoid arthritis, osteoarthritis and other autoimmune conditions.

[0137] In certain embodiments, compounds of the invention represented by
formula (1), are IFN-γ inhibitors and find use in therapies for
disorders associated with abnormal interleukin (IFN-γ) activity,
including: rheumatoid arthritis, osteoarthritis and other autoimmune
conditions.

[0138] In certain embodiments, compounds of the invention represented by
formula (1), down-regulate one or more gene selected from BCL2,
CEBPα, CEBPβ, CEBPδ, IL-1β, IL-6, cMyc, GBP-1,
MMP13 and MyD88 and find use in therapies for inflammatory disorders
including: Burkitt's lymphoma or Peutz-Jeghers syndrome.

[0139] According to an embodiment, compounds of the invention represented
by formula (1), down-regulate transcriptional targets of CREB such as
IL-1β in synovial cells and are useful for the treatment of an
inflammatory disorder mediated by CREB pathway.

[0140] According to an embodiment, the present invention provides a method
for monitoring drug response in a patient with an inflammatory disorder
treated with a compound of formula (1), comprising determining the
expression of one or more genes selected from CEBPα, CEBPβ,
CEBPδ, IL-1β, IL-6, GBP-1, MMP 13, MyD88, BCL2 and cMyc in a
test sample from the treated patient and comparing it to the expression
of the same one or more genes selected from CEBPα, CEBPβ,
CEBPδ, IL-1β, IL-6, GBP-1, MMP 13, MyD88, BCL2 and cMyc in a
test sample obtained from the patient before treatment with the compound
of formula (1) or in comparison with untreated controls.

[0141] In another embodiment, in the method of monitoring drug response, a
change of the expression of one or more genes selected from CEBPα,
CEBPβ, CEBPδ, IL-1β, IL-6, GBP-1, MMP 13, MyD88, BCL2 and
cMyc after treatment is indicative of a drug response.

[0142] In another embodiment, in the method of monitoring drug response
after the treatment with the compound of formula (1), the expression of
one or more genes selected from CEBPα, CEBPβ, CEBPδ,
IL-1β, IL-6, GBP-1, MMP 13, MyD88, BCL2 and cMyc is down-regulated.

[0144] According to an embodiment, compounds of the invention represented
by formula (1), find use in therapies for inflammatory disorders
including: rheumatoid arthritis and ulcerative colitis.

[0145] According to an embodiment, compounds of the invention represented
by formula (1), find use in the treatment of rheumatoid arthritis.

[0146] According to an embodiment, compounds of the invention represented
by formula (1), find use in the treatment of ulcerative colitis.

[0147] According to an embodiment, compounds of the invention represented
by formula (1), find use in the treatment of psoriasis.

[0148] According to an embodiment, the present invention provides a method
for the treatment of an inflammatory disorder mediated by one or more
cytokines selected from Tumor Necrosis Factor-alpha (TNF-α),
interferon-γ (IFN-γ), and interleukins such as IL-1β,
IL-2, IL-6, and IL-8 by administering to a mammal in need thereof a
therapeutically effective amount of one or more compound of formula (1).

[0150] According to another embodiment, the present invention provides a
method for the treatment of inflammatory disorders associated with
abnormal interleukin (IL-1β, IL-2, IL-6 and IL-8) including:
rheumatoid arthritis, osteoarthritis and other autoimmune conditions; by
administering to a mammal in need thereof a therapeutically effective
amount of one or more compound of formula (1).

[0151] According to another embodiment, the present invention provides a
method for the treatment of inflammatory disorders associated with
abnormal interleukin (IFN-γ) activity, including: rheumatoid
arthritis, osteoarthritis and other autoimmune conditions; by
administering to a mammal in need thereof a therapeutically effective
amount of one or more compound of formula (1).

[0153] According to an embodiment, the present invention provides a method
for the treatment of inflammatory disorders including: rheumatoid
arthritis and ulcerative colitis; by administering to a mammal in need
thereof a therapeutically effective amount of one or more compound of
formula (1).

[0154] According to another aspect of the present invention, there are
provided pharmaceutical compositions including a therapeutically
effective amount of one or more compound of formula (1) as active
ingredient and pharmaceutically acceptable carrier, useful in the
treatment of an inflammatory disorder mediated by one or more cytokines
selected from Tumor Necrosis Factor-alpha (TNF-α),
interferon-γ (IFN-γ) and interleukins such as IL-1β,
IL-2, IL-6 and IL-8.

[0155] According to another aspect of present invention, there are
provided methods of treatment of an inflammatory disorders mediated by
one or more cytokines selected from Tumor Necrosis Factor-alpha
(TNF-α), interferon-γ (IFN-γ) and interleukins such as
IL-1β, IL-2, IL-6 and IL-8 using these compositions as described
herein above.

[0156] According to another aspect of present invention there are provided
methods for manufacture of medicaments including one or more compounds of
formula (1), which are useful for the treatment of inflammatory disorders
mediated by one or more cytokines selected from Tumor Necrosis
Factor-alpha (TNF-α), interferon-γ (IFN-γ) and
interleukins such as IL-1β, IL-2, IL-6 and IL-8.

[0157] The pharmaceutical compositions according to the present invention
are prepared in a manner known per se and familiar to one skilled in the
art. Pharmaceutically acceptable inert inorganic and/or organic carriers
and/or additives can be used in addition to the compound(s) of the
formula (1), and/or its physiologically tolerable salts and/or its
prodrugs. For the production of pills, tablets, coated tablets and hard
gelatin capsules it is possible to use, for example, lactose, corn starch
or derivatives thereof, gum arabic, magnesia or glucose, etc. Carriers
for soft gelatin capsules and suppositories are, for example, fats,
waxes, natural or hardened oils, etc. Suitable carriers for the
production of solutions, for example injection solutions, or of emulsions
or syrups are, for example, water, physiological sodium chloride solution
or alcohols, for example, ethanol, propanol or glycerol, sugar solutions,
such as glucose solutions or mannitol solutions, or a mixture of the
various solvents which have been mentioned.

[0158] In addition to the active ingredients of the compound of formula
(1), and/or its physiologically acceptable salts and/or prodrugs and
carrier substances, the pharmaceutical compositions can contain additives
such as, for example, fillers, antioxidants, dispersants, emulsifiers,
defoamers, flavors, preservatives, solubilizers or colorants. The
pharmaceutical compositions of the present invention can also contain two
or more compounds of the formula (1) and/or its physiologically tolerable
salts and/or their prodrugs. Furthermore, in addition to at least one
compound of the formula (1), and/or its physiologically tolerable salts
and/or its prodrugs, the pharmaceutical compositions can also contain one
or more other therapeutically or prophylactically active ingredients.

[0159] The pharmaceutical compositions normally contain about 1 to 99%,
for example, about 5 to 70%, or about 10 to about 30% by weight of the
compounds of formula (1) or their physiologically tolerable salts or
their prodrugs. The amount of the active ingredient of formula (1),
and/or its physiologically tolerable salts and/or its prodrugs in the
pharmaceutical compositions can, for example, be from about 5 to 500 mg.
The dose of the compounds of this invention, which is to be administered,
can cover a wide range. The dose to be administered daily is to be
selected to suit the desired effect. A dosage of about 0.001 to 100
mg/kg/day of the compound of formula (1) or a prodrug thereof may be
administered per day. If required, higher or lower daily doses can also
be administered.

[0160] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of this invention can be varied so as to
obtain an amount of the active ingredient, which is effective to achieve
the desired therapeutic response for a particular patient, composition,
and mode of administration without being toxic to the patient.

[0161] The selected dosage level will depend upon a variety of factors
including the activity of the particular compound of the present
invention employed, the route of administration, the time of
administration, the rate of excretion of the particular compound being
employed, the duration of the treatment, other drugs, compounds and/or
materials used in combination with the particular compounds employed, the
age, sex, weight, condition, general health and prior medical history of
the patient being treated, and like factors well known in the medical
arts. The pharmaceutical compositions according to the present invention
can be administered orally, for example in the form of pills, tablets,
coated tablets, capsules, granules or elixirs. Administration, however,
can also be carried out rectally, for example in the form of
suppositories, or parenterally, for example intravenously,
intramuscularly or subcutaneously, in the form of injectable sterile
solutions or suspensions, or topically, for example in the form of
solutions or transdermal patches, or in other ways, for example in the
form of aerosols or nasal sprays.

[0162] It is understood that modifications that do not substantially
affect the activity of the various embodiments of this invention are
included within the invention disclosed herein.

b) Black soil samples were collected from crop fields near village
Hosalingpur, Bellary, Karnataka, India and were transferred into sterile
plastic bags. The samples were maintained at 4-8° C. c) Isolation
of actinomycetes from this soil:

b) The seed medium (40 mL) was distributed in Erlenmeyer flasks (500 mL)
and flasks were autoclaved at 121° C. for 30 min. The flasks were
cooled to room temperature and each flask was inoculated with a loopful
of the well-grown producing strain (culture no. PM0224355) on the slant
and was shaken on a rotary shaker for 70-74 h at 230-250 rpm at
30° C. (±1° C.) to obtain the seed culture. c)
Composition of the production medium:

d) The production medium (200 mL) was distributed in Erlenmeyer flasks
(1000 mL) and flasks were autoclaved at 121° C. for 30 min cooled
to 29° C.-30° C. and each flask was seeded with 5 mL of the
seed culture (as obtained in example 2 (b)). e) Fermentation parameters:

b) The seed medium (200 mL) was distributed in Erlenmeyer flasks (1000
mL) and flasks were autoclaved at 121° C. for 30 min. The flasks
were cooled to room temperature and each flask was inoculated with a
loopful of the well-grown producing strain (culture no. PM0224355) on the
slant and was shaken on a rotary shaker for 70-74 h at 230-250 rpm at
29° C.-30° C. to obtain the seed culture.

b) In fermenter (150 L), the above production medium (100 L) along with
desmophen (30 mL) as an antifoaming agent was sterilized in situ for 30
min at 121° C., was cooled to 29° C.-30° C. and was
seeded with 2.5-3.5 L of the seed culture (as obtained in step 1(b),
example 4). c) Fermentation parameters:

[0251] Crude ethyl acetate extract (as obtained in step 1, example 5) was
purified by column chromatography (silica gel, methanol in chloroform).
The fractions were monitored by TLC (silica gel, chloroform-methanol 9:1,
detection: 254 nm) using concanamycin A as a reference standard. The
fraction which was eluted with 3% methanol in chloroform, was
concentrated to obtain extract enriched with concanamycins (5 g). The
extract enriched with concanamycins was dissolved in methanol, kept at
4° C. for 10-12 h, and was filtered to obtain a powder (Yield: 0.6
g) which was identified as containing mixture of concanamycin A and
concanamycin C by LCMS (molecular weight 865 and 822). This is referred
to as concanamycin crude.

[0253] NaOH solution in methanol (0.03M) was added to the powder (100 mg)
(as obtained in step 2, example 5) at 10° C. and the mixture was
stirred for 20 min. The reaction mixture was neutralized using HCl (1 N)
and was extracted with ethyl acetate (3×10 mL). The organic layer
was washed with water, dried over sodium sulphate and was concentrated.
The crude product was purified by column chromatography (silica gel, 30%
ethyl acetate in petroleum ether) to obtain the title compound. Yield: 55
mg.

[0259] The compound of example 6 (12 mg) was dissolved in the mixture of
pyridine (1 mL) and ethanol (1 mL) and was reacted with hydroxylamine
hydrochloride (3.17 mg) under nitrogen at 25° C. for 4 h. Water
was added to the reaction mixture and the reaction mixture was extracted
with ethyl acetate (3×5 mL). The organic layer was washed with
water, dried over sodium sulphate and was concentrated. The crude product
was purified by column chromatography (silica gel, 40% ethyl acetate in
petroleum ether) to obtain the title compound. Yield: 10 mg. HPLC: 99.2%
pure, retention time 25.2 min, [RP-18 (4 mm×250 mm) column, 2-100%
gradient of acetonitrile in water over 35 min at 25° C.,
detection: 220 nm]; MS: m/e 689;

[0264] The compound of example 6 (10 mg) was dissolved in the mixture of
pyridine (500 μL) and ethanol (500 μL) and was reacted with
methoxyamine hydrochloride (6.5 mg) under nitrogen at 25° C. for 4
h. Water was added to the reaction mixture and the reaction mixture was
extracted with ethyl acetate (3×5 mL). The organic layer was washed
with water, dried over sodium sulphate and was concentrated. The crude
product was purified by preparative HPLC [Eurospere-100, C18 column (250
mm×8 mm), mobile phase: acetonitrile-water (1:1 isocratic)] to
obtain the title compound. Yield: 6.5 mg; MS: m/e: 703.

[0266] The compound of example 6 (10 mg) was dissolved in the mixture of
pyridine (300 μL) and ethanol (700 μL) was reacted with benzyloxy
amine hydrochloride (8.4 mg) in pyridine under nitrogen at 25° C.
for 4 h. Water was added to the reaction mixture and the reaction mixture
was extracted with ethyl acetate (3×5 mL). The organic layer was
washed with water, dried over sodium sulphate and was concentrated. The
crude product was purified by preparative HPLC [Eurospere-100, C18 column
(250 mm×8 mm), mobile phase: acetonitrile-water (1:1 isocratic)] to
obtain the title compound. Yield: 7.2 mg; MS: m/e: 779.

[0280] The compound of example 6 (3 mg) was dissolved in acetone (1 mL)
and was reacted with KOH (1 mg) and tosyl chloride (1.8 mg) under
nitrogen at 0° C. for 2 h. Stirring was continued for 1 h at room
temperature. Cold water was added to the reaction mixture and the
reaction mixture was extracted with ethyl acetate (3×5 mL). The
organic layer was washed with water, dried over sodium sulphate, and was
concentrated. The crude product was purified by preparative TLC [silica
gel, mobile phase: ethyl acetate-hexane (1:1)] to obtain the title
compound. Yield: 0.7 mg. MS: m/e: 689.

[0282] The compound of example 6 (5 mg) was dissolved in tetrahydrofuran
(1 mL) and was subjected to reaction with sodium borohydride (0.56 mg)
and cerium(III) chloride (CeCl3) (0.9 mg) under nitrogen at
0° C. for 20 min, then was stirred for 20 min at room temperature.
Cold water was added to the reaction mixture and it was extracted with
ethyl acetate (3×5 mL). The organic layer was washed with water,
dried over sodium sulphate and was concentrated. The crude product was
purified by preparative TLC [silica gel, mobile phase: hexane-ethyl
acetate (1:1)]. to obtain the title compound. Yield: 2.7 mg; MS: m/e 676.

Pharmacology

[0283] The efficacy of the compounds of formula (1) and formulations, in
inhibiting the activity of one or more cytokines selected from
TNF-α, interferon-γ (IFN-γ) and interleukins
(IL-1β, IL-2, IL-6, and IL-8), was determined by pharmacological
assays well known in the art and are described below.

Example 18

Screening in LPS Stimulated THP-1 Cells

[0284] IL-6 (BD Biosciences, USA) production by LPS (Escherchia coli
0127:B8, Sigma, USA) in THP-1 cells (ATCC number: TIB202) was designed as
in reference, Journal of Immunology, 151, 5631-5638, (1993), the
disclosure of which is incorporated by reference for the teaching of the
assay.

[0285] THP-1 cells were cultured in RPMI 1640 culture medium (Gibco BRL,
UK) containing 100 U/mL penicillin and 100 mg/mL streptomycin,
(100× solution, Sigma, USA) containing 10% FBS (JRH Biosciences,
USA). 25,000 cells were seeded per well in 96-well plate (Nunc, USA). The
cells were differentiated with PMA (Sigma, USA, prepared as 100 μg/mL
stock in RPMI and was diluted to 5 ng/mL). The test compound (prepared as
20 mM stock in DMSO and diluted with DMSO to achieve the following final
concentrations in the assay: 100, 10, 1, 0.1, 0.01, 0.001 and 0.0001
μM) or vehicle (0.5% DMSO) were added to the cells and the cells were
incubated for 30 min at 37° C. LPS (Sigma, USA, prepared as 1
mg/mL stock in PBS) was added to achieve a final concentration of 1
μg/mL. Plates were incubated at 37° C. for 24 h at 5% CO2.
Supernatants were harvested, and assayed for TNF-α and IL-6 by
ELISA as described by the manufacturer (BD Biosciences, USA). Percent
inhibition of cytokine release compared to the control was calculated.
The IC50 values were calculated by a nonlinear regression method.
Results obtained are summarized in Table 1.

[0289] The assay method was designed as in references, The Journal of
Immunology, 153, 1-9, (1994), and Clinical and Diagnostic Laboratory
Immunology, 7, 687-692, (2000), the disclosure of which is incorporated
by reference for the teaching of the assay.

Step 1

[0290] Isolation of hPBMCs

[0291] hPBMCs were obtained from healthy donors by centrifugation of
heparinized venous blood over Ficoll/Hypaque solution (Histopaque-1077,
Sigma, USA). Isolated hPBMCs were suspended in RPMI 1640 culture medium
supplemented with 10% FBS and seeded at a density of 50,000 cells/well in
a 96-well plate (Nunc, USA). The cells were incubated at 37° C.,
5% CO2 for a period of 24 h. These cells were used for the
lymphocyte proliferation as well as cytokine release assay.

Step 2

Lymphocyte Proliferation Assay

[0292] The plated cells were treated with different concentrations of the
test compound (prepared as 20 mM stock in DMSO and diluted with DMSO to
achieve the following final concentrations in the assay: 100, 10, 1, 0.1,
0.01, 0.001 and 0.0001 μM) and were incubated for 30 min. The cells
were then stimulated with 5 ng/mL PMA (Sigma, USA, prepared as 100
μg/mL stock in RPMI and was diluted to 5 ng/mL) and 5 μg/mL PHA
(Sigma, USA, prepared as a 1 mg/mL stock in RPMI). The plates were
incubated at 37° C., 5% CO2 for 48 h. The cells were treated
overnight with 0.1 μCi of tritiated thymidine per well (obtained from
BARC, India; prepared as stock of 1 mCi/mL) and was diluted in KRPH
buffer to 10 μCi/mL and 20 μL and was added per well to obtain a
final concentration 0.1 μCi/well) and at the end of 48 h, the
anti-proliferative effect of the compound was measured using the
following formula:

[0294] The plated cells were treated with different concentrations of the
test compound (prepared as mM stock in DMSO and diluted with DMSO to
achieve the following final concentrations in the assay: 100, 10, 1, 0.1,
0.01, 0.001, 0.0001, 0.00001 and 0.000001 μM) and incubated for 30
min. The cells were then stimulated with PHA (prepared as 1 mg/mL stock
in RPMI 1640 culture medium and used at a final concentration of 5
μg/mL). FK506 was used as a standard. The plates were incubated at
37° C., 5% CO2 for 48 h. The cytokines in the supernatant
collected were detected using ELISA kit (BD biosciences, USA). The
cytokines evaluated in the assay were TNF-α, IL-2, IL-6, and
IFN-γ. Results obtained are summarized in Table 3. and Table 4

[0297] Representative compounds of the present invention significantly
blocked the production of the Th1 cytokines; namely IL-2, IFN-γ and
TNF-α and also inhibited IL-6 production.

Example 20

Human Monocyte Assay

[0298] The assay was designed as in reference, Physiological Research, 52,
593-598, (2003), the disclosure of which is incorporated by reference for
the teaching of the assay.

[0299] The objective of the assay is to determine whether compounds of the
present invention--mediated inhibition of LPS-induced cytokines from
monocytic THP-1 cell line translates to physiologically relevant human
cells. Accordingly, the effect of compounds of the present invention on
LPS--induced cytokine production from freshly isolated human monocytes
was ascertained.

[0300] Peripheral blood was collected from healthy donors into potassium
EDTA vacutainer tubes (BD Biosciences, USA). hPBMCs were isolated using
density gradient separation (Histopaque-1077; Sigma, USA) and suspended
in assay medium which is RPMI culture medium (Sigma, USA) containing 10%
heat inactivated FBS (JRH Biosciences, Australia), 100 U/mL penicillin
(Sigma, USA) and 100 mg/mL streptomycin (Sigma, USA). Monocytes in the
hPBMCs were counted using a Coulter Counter following which the cells
were resuspended at 2×105 monocytes/mL of assay medium. A cell
suspension containing 2×104 monocytes was aliquoted per well
of a 96-well plate (Nunc, USA). Subsequently, the hPBMCs were incubated
for 4-5 h at 37° C., 5% CO2. During the incubation, the
monocytes adhered to the bottom of 96-well plate. Following the
incubation, the non-adherent lymphocytes were washed and assay medium was
added to adherent monocytes. After 48 h of incubation at 37° C.,
5% CO2, monocytes were pre-treated with various concentrations of
test compound (prepared as 20 mM stock in DMSO; 1 μL of 20×
concentrated solution of test compound was dissolved in 200 μL cell
suspension to achieve a final concentration of 0.03, 0.1, 0.3, 1, 3, 10,
30 and 100 μM) or vehicle (0.5% DMSO) or 10 μM dexamethasone
(standard IL-6 and TNF-α inhibitor, Sigma, USA) for 30 min at
37° C., 5% CO2 and stimulated with 1 μg/mL LPS (Escherchia
coli 0111:B4, Sigma, USA). The cells were then incubated for 5 h at
37° C., 5% CO2 following which supernatants were collected,
stored at -70° C. and were assayed later for IL-6, and TNF-α
by ELISA (OptiEIA ELISA sets, BD Biosciences, USA). The IC50 values
were calculated by a nonlinear regression method using Graph Pad software
(Prism 3.03).

[0301] In all experiments, a parallel plate was run to ascertain the
toxicity of test compounds. A cell proliferation assay kit (Promega Life
Sciences, USA), containing MTS tetrazolium salt, was used to assess the
viability of the monocytes. Viable cells reduce MTS to form a colored
product. The protocol used was as per the manufacturer's instructions and
as detailed in the following reference, Am J Physiol Cell Physiol., 285,
C813-C822, (2003). A MTS/PMS stock solution was prepared by mixing 2 mL
MTS with 100 μL PMS (Sigma, USA) (Stock solution of MTS was prepared
as follows: 1 gm of MTS was dissolved in 500 mL of DPBS with calcium and
magnesium. Subsequently, the solution was filtered using 0.2 μM filter
(Nunc, USA). Aliquots were stored at -20° C. Stock solution of PMS
was prepared as follows: 18.4 mg of PMS was dissolved in 20 mL of DPBS
with calcium and magnesium. Subsequently, the solution was filtered using
0.2 μM filter sterilized using 0.2 μM syringe filter (Millipore,
USA). Aliquots were stored at -20° C. Subsequently, 40 μL of
the above mentioned MTS/PMS solution was added to each well of a 96-well
plate containing the 2×104 monocytes (resuspended in a volume
of 200 μL). After 5 h incubation at 37° C., 5% CO2, the
absorbance of the fluid in each well was determined at 490 nm using the
Microwell plate spectrophotometer. The results are summarized in Table 6.

[0302] Representative compounds of the present invention inhibited
LPS-induced production of IL-6 and TNF-α

Example 21

Synovial Tissue Assay

[0303] The ability of compounds of the present invention to inhibit
spontaneous production of cytokines from freshly isolated human synovial
tissue cells was designed as in reference, Lancet, 29, 244-247, (1989)
the disclosure of which is incorporated by reference for the teaching of
the assay.

[0304] Synovial tissue was obtained from rheumatoid arthritis patients
undergoing knee replacement surgery. The tissue was minced into small
pieces and digested in RPMI 1640 culture medium (JRH Biosciences,
Australia) containing 100 U/mL penicillin-G, 100 μg/mL streptomycin,
50 ng/mL amphotericin B (Gibco, USA), 1.33 mg/mL collagenase Type I
(Worthington Biochemical Corporation, USA), 0.5 μg/mL DNAse Type I
(Sigma, USA) and 8.33 U/mL heparin (Biological E. Limited, India) for 3 h
at 37° C., 5% CO2. The digested tissue was filtered through a
membrane (mesh size 70 micron; Sigma, USA). Subsequently, the cells were
washed 3 times with RPMI 1640 culture medium and resuspended in complete
medium (RPMI 1640 culture medium supplemented with 5% FBS and 5% human
serum-AB+(Sigma, USA) at a concentration of 1×106 cells/mL.
The viability of synovial cells was determined by trypan blue dye
exclusion and was uniformly ≧98%. For the experiment, 100 μL of
cell suspension was added to the wells of a 96-well culture plate (Nunc,
USA). Following cell plating, 100 μL of the culture medium and 1 μL
of various concentrations of the test compound (test compound was
dissolved in DMSO to obtain a stock solution of 20 mM. 1 μL of
20× concentrated solution of test compound was dissolved in 200
μL cell suspension to achieve a final concentration of 0.03, 0.1, 0.3,
1, 3, 10, 30 and 100 μM in the assay) were added to the cells. The
final concentration of DMSO was adjusted to 0.5%. The vehicle (0.5% DMSO)
was used as control. The plates were incubated for 16 h at 37° C.,
5% CO2. Subsequently, the supernatants were harvested and stored at
-70° C. The amounts of TNF-, IL-6 and IL-8 in the supernatants
were assayed using OptiEIA ELISA sets (BD BioSciences, USA). The protocol
followed was as per manufacturers instructions. The IC50 values were
calculated by a nonlinear regression method using the GraphPad software
(Prism 3.03).

Result:

[0305] Compound of example 7 inhibited the spontaneous production of IL-6,
TNF-α, and IL-8 from freshly isolated synovial tissue cells from
rheumatoid arthritis patients. The IC50 of TNF-α, IL-6, and
IL-8 inhibition were 19, 0.3 and 1.3 μM respectively. The IC50
for inhibition of TNF-α and IL-6 from synovial tissue cells were
comparable to the IC50 values obtained in the human monocyte assay.

Example 22

[0306] hPBMC Membrane--Monocyte Contact Assays

[0307] The assay was designed as in reference, Immunology Letters, 117,
114-118, (2008), the disclosure of which is incorporated by reference for
the teaching of the assay. Activated T cell contact-mediated monocyte
activation, leading to the production of proinflammatory cytokines (e.g.,
TNF-α, IL-6), contributes to the pathogenesis of chronic
inflammatory diseases including rheumatoid arthritis. The objective of
this assay is to investigate whether compounds of the present invention
inhibit anti-CD3/anti-CD28 activated hPBMC-mediated TNF-α and IL-6
production from monocytes.

[0309] The plates were incubated overnight at 4° C. under sterile
conditions. After 24 hours, plates were washed once with sterile PBS
(without calcium/magnesium), following which, the plates were incubated
with anti-CD3 (5 μg/mL; R&D Systems, USA) and anti-CD28 (1 g/mL; R&D
Systems, USA) cocktail in sterile PBS for 3 h. After 3 h, the plates were
washed once with PBS, and were used for hPBMC stimulation.

Step 2

[0310] hPBMC Membrane Preparation

[0311] The hPBMC membranes were prepared in a manner similar to the
preparation of T-cell membranes as described in Immunology Letters, 15,
117(1):114-118, (2008).

[0313] Subsequently, the hPBMCs in the plate were incubated at 37°
C., 5% CO2 for 24 h.

[0314] Following incubation, hPBMCs in separate wells of the 6-well plate
were harvested, pooled together, and centrifuged. The supernatants were
collected and stored at -70° C. for later analysis for cytokine
production as confirmation for anti-CD3/anti-CD28 activation of hPBMCs.
The pelleted hPBMCs were washed twice in cold PBS and resuspended in
Tris-HCl buffer [PBS containing 50 mM Tris-HCl, pH 7.4; 1 mM EDTA; and
protease inhibitor cocktail (Roche, USA)]. The activated/unactivated
hPBMCs were broken down by homogenization (Polytron PT 3100 homogenizer)
at 10,000 to 12,000 rpm for 1 min, the nucleus fraction was obtained by
centrifugation at 4000×g for 15 min, and the supernatant was
centrifuged for 45 min at 48,000×g. The pellet of hPBMC membranes
was resuspended in lysis buffer (Sigma, USA) and the protein
concentration was determined by the method of Bradford (Sigma, USA).

Step 3

[0315] hPBMC Membrane-Monocyte Contact Bioassay:

[0316] 100 μL monocytes per well at 5×105 cells/mL were
added to the 96-well plate (Nunc, USA) and cultured for 48 h at
37° C. Thereafter, supernatants were removed and the cells were
further incubated with either unstimulated or anti-CD3/anti-CD28
stimulated hPBMC membranes (0.5-1 μg/mL) or with LPS as a positive
control (the stock solution of LPS (1 mg/mL) was prepared in complete
medium RPMI 1640 culture medium containing 10% FBS, 100 U/mL penicillin
and 100 mg/mL streptomycin). LPS was diluted in complete medium and a
20× solution of LPS was added such that the final concentration of
LPS was 1 g/mL in each well containing monocytes. To determine the effect
of compounds of the present invention, monocytes were pre-treated with
various concentrations of test compound (prepared as 20 mM stock in DMSO.
1 μL of 20× concentrated solution of test compound was dissolved
in 200 μL cell suspension to achieve a final concentration of 0.03,
0.1, 0.3, 1, 3, 10, 30 and 100 μM of test compound in the assay) or
0.5% DMSO (vehicle control) for 30 min at 37° C. Stimulated hPBMC
membranes were then added to the culture. Supernatants were collected
after 24 h and TNF-α and IL-6 production was measured using OptiEIA
ELISA sets, (BD BioSciences, USA). The protocol followed was as per
manufacturers instructions. In each experiment, supernatants from
cultures of monocytes alone without hPBMC cell membranes or hPBMC
membranes without monocytes were also collected as negative controls.

Result:

[0317] Compound of example 7 inhibited the activated hPBMC mediated IL-6
production, 66% at 0.3 μM and 100% at 0.5 μM, but did not inhibit
TNF-α production from monocytes.

[0319] Assays were carried out to select for non inhibitors of p38 MAPK
since p38 MAPK inhibitors have demonstrated hepatotoxicity in clinical
trials. The method for identifying inhibitors of p38 MAPK was designed as
in reference, Journal of Lipid Research, 40, 1911-1919, (1999), the
disclosure of which is incorporated by reference for the teaching of the
assay.

[0320] Human Jurkat T-cells (ATCC number: TIB-152, clone E6-1, USA) were
cultured in culture medium (RPMI 1640 culture medium supplemented with
10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin) at 37°
C., 5% CO2. Culture medium was changed every 2-3 days and always a
day prior to the experiment. On the day of the experiment, Jurkat cells
were pre-treated with vehicle or test compound at 3 μM, 10 times the
IC50 value for IL-6 inhibition in human monocyte assay, for 1 h at
37° C. Subsequently, the cells were stimulated with anisomycin (10
μg/mL; Sigma, USA) for 30 min SB 203580 (1 PM; Sigma, USA) was used as
a standard. The sample preparation of the test compound, anisomycin and
SB 203580 is as follows: a stock solution (20 mM) of the test compound
was prepared in DMSO. All subsequent dilutions of the compound were
performed using DMSO. 1 μL of appropriate concentration of the
compound was added to the cell suspension to achieve the desired final
concentration in the well.

[0323] Representative compound of the present invention did not inhibit
p38 MAP kinase.

Example 24

Gene Expression Profile Using RTQ-PCR

[0324] The effect of the compound of example 7 was measured in stimulated
untreated cells from the monocytic cell line (THP-1), human monocytes and
synovial cells from rheumatoid arthritis patient. The effect of the
compound in the THP-1 cell line was measured in terms of gene expression
and was expressed as fold changes as compared to the cell stimulated
control with no drug treatment.

[0325] THP-1 cells, human monocytes and synovial cells were treated with
compound of example 7 or vehicle (0.5% DMSO). Total RNA isolation using a
commercial RNA extraction kit (Qiagen Corporation, Germany) The
first-strand cDNA was synthesized from total RNA using first strand cDNA
synthesis kit from Invitrogen Corporation (California, USA). This was
followed by real time quantitative polymerase chain reaction (RTQ PCR)
using gene specific primers and standard thermal program of initial
denaturation at 95° C. for 5 min and cycles of 95° C. for
10 seconds, followed by 60° C. for 30 seconds (Realplex PCR
machine from Eppendorf, Germany). Quantitative measurement of products
made during PCR cycles was normalized against a housekeeping gene (Actin)
and was used to measure the gene expression as fold changes as compared
to respective control. The results are summarized in Table 7A and Table
7B

[0328] Animals used in the experiments were housed and cared for, in
accordance with the Guidelines in force published by CPCSEA (Committee
for the Purpose of Control and Supervision of Experiments on Animals),
Tamil Nadu, India. Procedures using laboratory animals were approved by
the IAEC (Institutional Animal Ethics Committee) of Piramal Life Sciences
Limited, Goregaon, Mumbai, India.

Example 25

DSS Induced Murine Model of Acute Colitis

[0329] The assay was designed as in references, Laboratory Investigation,
80, 1541, (2000), and Faseb Journal, 19:792, (2005), the disclosure of
which is incorporated by reference for the teaching of the assay.

[0331] Experimental colitis was induced in mice by replacing drinking
water with 3% (w/v) DSS (molecular weight 36,000-50,000, MP Biomedicals
Inc., USA) solution. This solution was prepared in water, freshly every
alternate day and was made available to the experimental animals ad
libitum, from day 0 to day 10. A batch of six naive animals received
water instead of DSS during this period.

Step 2

Treatment:

[0332] The animals were weighed every day and the record of body weights
was maintained. The suspension of test compound (was prepared at a
concentration of 0.05 mg/mL, in 0.5% (w/v) CMC after mixing with minimum
quantity of Tween 80 necessary to wet the compound, and was administered
orally, daily once to the animals at a volume of 10 mL/kg (dose=0.5
mg/kg). This treatment was initiated on day 6 and continued up to day 10.
During this period, DSS control animals and naive animals received CMC
(mixed in the same proportion with 100 μL of Tween 80) at a dose of 10
mL/kg, daily once.

Step 3

Terminal Sacrifice:

[0333] On day 11, the animals were sacrificed, blood was collected in
heparinized tubes and the following parameters were studied; [0334] 1.
Rectal bleeding/blood in faeces [0335] 2. Fecal consistency [0336] 3.
Bleeding in colon [0337] 4. Colon weight [0338] 5. Colon length [0339] 6.
Change in body weight on day 11 from that of day 0 [0340] 7. Blood
hemoglobin concentration

[0341] The data are represented in the following tables (Table 8, Table 9
and Table 10) as quantifiable parameters, descriptive parameters and
actual scores. The descriptive parameters are represented by DAI. DAI is
a research tool used to quantify the symptoms of the colitic animals. DAI
is used in order to define response of the treatment or remission of the
disease. In order to achieve this, various factors are studied. Some of
these factors are quantifiable (e.g. change in body weight during the
experimental period, colon length, blood hemoglobin concentration) and
hence can be directly used to assess the beneficial effect of the
treatment; others are just descriptive (e.g. blood in colon, rectal
bleeding, fecal consistency) and are scored according to the severity of
the disease. DAI is the sum of the scores of all factors. Results are
summarized in Tables 8, 9 and 10.

[0342] Compound of example 7, when administered orally at a dose of 0.5
mg/kg to the experimental animals (mice) with DSS induced colitis,
reduces the severity of colitis.

Step 4

Histopathology

[0343] On the last day of compound treatment (compound of example 7, 0.5
mg/kg, p.o., b.i.d) the animals were euthanized humanely, blood samples
were collected and the colon was excised. Anterior part of the colon was
washed with normal saline to remove fecal material and then fixed in 10%
NBF (Neutral buffered formalin). After 10 days of fixation the colon
specimens were trimmed and processed overnight using automated tissue
processor. Following day the specimens were blocked in paraffin and
exposed to cold shock overnight at -18° C. Sections (5μ) were
made from cross section of the colonic lumen and stained with routine
Hematoxyllin (Sigma, USA) & Eosin (Loba Chemie, India) and mounted
permanently. Slides were dried for 24 h and then graded histologically.
The results were expressed as histological scores. Histological analysis
was based on various parameters like presence of inflammatory cells,
erosions, crypt destruction, edema and overall architectural changes
graded on a score of 0 to 3, wherein 0 corresponds to absence, 1
corresponds to changes in 25% of the circumference of the colonic lumen,
2 corresponds to up to 50% and 3 corresponds to more than 50% of colonic
circumference getting affected. All the scores were summed up to arrive
to a total histological score for each section. Two random sections were
graded and mean was calculated.

Result:

[0344] Group mean histological scores were 7.50+0.96 for DSS control and
5.33+0.99 for mice treated with compound of example 7.

[0346] The experiment was designed as in reference, J. Experimental
Medicine, 162, 637-646 (1985), the disclosure of which is incorporated by
reference for the teaching of the experiment.

[0347] Male DBA/1J mice with body weight range of 18-22 g, aged 8-10 weeks
were immunized with an emulsion equivalent to 200 μg of type II
collagen (Elastin products, USA) in Freund's Complete Adjuvant (Sigma,
USA), injected intradermally at the base of the tail. The animals were
boosted with 200 μg of freshly prepared typeII collagen emulsion
emulsified in Freund's Complete Adjuant (Sigma, USA) on day 21. A group
of naive mice was also maintained alongside. Naive animals are the
animals which are neither immunized for induction of arthritis nor do
they receive any treatment. This group is maintained to take care of the
normal changes in the paw thickness with age.

[0348] From day 23, mice were examined daily once for the signs of
rheumatoid arthritis, using the articular index and paw thickness as
parameters. Articular index scoring was performed employing the following
criteria:

FORELIMBS: SCALE 0-3

[0349] 0: No redness or swelling [0350] 1: Redness, but no swelling
[0351] 2: Redness and swelling of the paw [0352] 3: Redness and severe
swelling of the paw Hind limbs: Scale 0-4 [0353] 0: No redness or
swelling [0354] 1: Redness and mild swelling of paw [0355] 2: Redness and
moderate swelling of paw and/or swelling of at least one of the digits
[0356] 3: Redness and moderate/severe swelling of paw, swelling of ankle
joint and/or swelling of one or more digits [0357] 4: Redness and severe
swelling of paw, digits and ankle joint, with joint stiffness Mice with a
minimum hind paw score of 2, of even one paw, were inducted into the
study. Mice were randomized into the various study groups, each group
having at least eight animals, and were administered the vehicle (0.5%
CMC, 10 mL/kg p.o. and s.c. twice daily), the test compound (5 mg/kg,
p.o. and s.c., twice daily) and standard compound Enbrel (Wyeth Limited,
UK), 3 mg/kg, s.c., once daily. The test compound was administered as a
suspension in CMC. The requisite quantity of the compound was accurately
weighed and was hand-pulverized using pestle-mortar. After mixing with
minimum quantity of Tween 80 necessary to wet the compound, requisite
quantity of 0.5% CMC solution was added and the compound was ground with
CMC till the uniform suspension was obtained. Standard compound `Enbrel`
was used as an aqueous solution. The dosing of the compounds was done for
12 continuous days.

[0358] The following parameters were observed and recorded daily, [0359]
1. Body weight [0360] 2. Articular index [0361] 3. Paw thickness of hind
limbs only, in mm using a tension free calipers [0362] 4. Any significant
observation regarding the condition of the animals.

[0363] On 13th day morning, 1 h after the compound treatment, the
animals were sacrificed, blood withdrawn, and plasma collected for drug
level analyses. Also, the hind limbs of all the animals were preserved
for histopathological evaluations.

Result:

[0364] Compound of example 7 at a dose of 5.0 mg/kg as a CMC suspension,
administered subcutaneously twice daily to the mice with collagen induced
arthritis, for 12 continuous days, reduced the severity of arthritis. The
benefit is equal to that achieved with Enbrel treatment (3 mg/kg, s.c.,
once daily).

Histopathology:

[0365] Histological score of compound of the section of paws of mice
treated with the compound of example 7 (5 mg/kg, s.c., n=10) was
3.6+1.54, Enbrel (3 mg/kg, s.c., n=6) treated mice had a score of
5.8+0.95 and that of vehicle control (n=7) was 15.14+1.0.

Observations:

[0366] The vehicle control group animals showed complete destruction of
joint architecture accompanied by severe hyperplasia of synovium and
pannus formation. Animals treated with compound of example 7 showed
protection against arthritic changes with absence of hyperplasia of
synovium.

[0367] The experiment was performed in DBA/1J mice, in which arthritis was
developed by injection of collagen emulsion, as described in Example 26.

[0368] The animals were divided into two groups, viz. a control group and
a test compound treated group, having 6 animals each. The clear solution
of test compound was prepared in 100% dimethyl sulfoxide (DMSO) and then
DMSO concentration was brought down to 25% by addition of appropriate
quantities of ethanol and polyethylene glycol 400 (PEG 400), so that the
proportion of each solvent in a final solution v/v was
25:15:60::DMSO:EtOH:PEG-400. By this method absolutely clear solution of
the compound having a final concentration of 40 mg/mL was obtained. This
solution was filtered through 0.2μ filter and was filled in osmotic
pumps (Alzet micro-osmotic pump model 1002). The delivery rate of this
model of pump is 0.25 μL per hour and it remains functional for 14
days.

[0369] These pumps were then implanted sub-cutaneously in the animals of
the test compound treated group (240 μg/mouse/24 h). The pumps filled
with blank solvent were implanted sub-cutaneously in the animals of
control group. Thereafter, their paws were scored for arthritic indices,
in addition to measurement of thickness, daily once. After 14 days, the
pumps were replaced with freshly filled pumps and the experiment was
continued for next 12 days (total 26 days).

Result:

[0370] The compound 7 of present invention, when sub-cutaneously
administered in the arthritic animals by means of the osmotic pumps
reduces severity of arthritis by the reductions in the arthritic scores
and paw thickness, when compared to the control group of animals.

Conclusion:

[0371] Compound 7 of present invention is efficacious in reducing the
severity of arthritis when administered sub-cutaneously.